Plant
What motivates us to delve into the realm of plant synthetic biology? Let's start by thinking about the most fundamental human needs.
With the rapid economic and social development, on the one hand, people's pace of life is accelerating, and bad lifestyle and dietary habits are gradually increasing, making the body in a long-term sub-healthy state, and the concern and demand for health is naturally becoming more and more urgent. Environmental pollution, food safety and other issues are becoming more and more prominent, bringing potential threats to people's health, further stimulating people's desire for health solutions. On the other hand, with the ever-changing environment, it brings many challenges to agricultural production and so on. And plant synthetic biology provides us with more solutions.
In this synthetic biology research, we used cucumber as a plant specimen to explore the potential of flavonoid synthesis using its own secondary metabolite “processing factory”, the epidermal trichome.
During the critical period of cucumber epidermal trichome development, the key flavonoid synthesizing genes were expressed in large quantities in specific epidermal trichome. Using CsTBH, a key gene controlling the development of epidermal trichome, and CsPAL, a key rate-limiting enzyme for flavonoid synthesis, as the design unit, we confirmed that CsTBH has the ability to activate the expression of CsPAL through experimental verification. We confirmed that CsTBH has the ability to activate the expression of CsPAL. Through the co-expression of CsTBH and CsPAL, we succeeded in increasing the content of flavonoids in the epidermis of cucumber.
The present study provides important theoretical support for the advancement of cucumber epidermal trichome-regulated metabolites, as well as a technical reference for the regulation of its metabolites in other plants. More importantly, utilizing this synthetic biology theory provides practical evidence for future modification of plants to produce more useful metabolites.
The epidermal trichome of several plants, such as Cannabis sativa and Artemisia annua, have been used to increase the production of cannabinoids and artemisinins (Hancock, et al., 2024; Li et al., 2024). However, so far, the key genes that regulate the initiation and development of epidermal trichome in Cannabis sativa and Artemisia annua are still unclear. In addition, the general public is poorly familiar with plants such as Cannabis sativa and Artemisia annua. Therefore, it is necessary to find a plant that is familiar to the general public and at the same time can be utilized to carry out synthetic biology research and popularize educational knowledge of epidermal trichome.
We chose cucumber as our plant sample because cucumber is the most widely distributed vegetable crop in the world and the most common food that people eat three meals a day, which is widespread and universal. At the same time, cucumber has its own easily observable epidermal trichome.
More importantly, in terms of scientific research, the initiation of epidermal trichome in cucumber is relatively cutting-edge, with the cloning of one of the most critical initiation genes, TINY BRANCHED trichome (CsTBH), and the production of metabolites, such as flavonoids, which are beneficial for improving resistance to ultraviolet rays, aphids, etc. (Feng et al., 2023; Zhang et al., 2021), suggesting that cucumber has a strong resistance to ultraviolet rays and aphids (Feng et al., 2023; Zhang et al., 2021). 2021), implying that the epidermal trichome of cucumber can also serve as an important organ for metabolite production, which is feasible for research. Furthermore, our strong familiarity with cucumber makes it convenient to carry out basic research and experimental materials. In addition, the cultivation technology of cucumber is relatively mature, and the cucumber can be well adapted to meet our research needs in the greenhouse environment for fine-tuning experiments. Moreover, the growth cycle of cucumber is relatively short, and it can complete the process from planting to harvesting in a relatively short time, which allows us to complete the experimental validation within a limited time and improve the research efficiency. From the perspective of economic cost, cucumber, as a common vegetable crop, has a relatively low cost of seeds, planting substrate and related planting equipment, which largely reduces our research cost. Compared with some rare plants or plants that require special growing conditions, cucumber is more economically feasible.
In order to increase the flavonoids in the fruit spines of cucumber epidermal trichome, CsTBH, a key gene controlling the morphogenesis of epidermal trichome, was screened and validated, and it was found that CsTBH stimulated the expression of PAL, a precursor of flavonoid synthesis, and then a CsTBH - CsPAL co-expression module was designed, which allowed the metabolite to be expressed only in fruit spines, providing a new idea of realizing the expression of a specific metabolite at a specific site. specific sites, providing a new idea to realize the expression of specific metabolites in specific sites.
Innovative correlation between the development of the epidermal trichome, a plant organ, and the production of specific metabolites, flavonoids. By ensuring the construction of a “metabolic factory” of epidermal trichome (CsTBH) and the control of flavonoid production (CsPAL as a master switch), and by establishing direct interactions between CsTBH and CsPAL, a new chassis screening technology has been developed for the direct regulation of modular crop programs. This is an innovative attempt to tightly integrate different levels of plant physiological processes.
In order to effectively produce flavonoids in cucumber epidermal trichomes, the overexpression vector PCY - 35S - His was introduced, and PCY - CsTBH overexpression vector and PCY - CsPAL overexpression vector were constructed to complete the chassis design. Meanwhile, the CsPAL promoter activated by CsTBH was constructed by using yeast single-hybrid vector PLacZi, and the precise regulation of gene expression and metabolite production was realized through the construction of multiple vectors with different functions.
comprehensive validation methods were adopted in validating the overexpression products of CsTBH and CsPAL. Using Pseudomonas aeruginosa to infest cucumber cotyledons for genetic transformation test, observing the color change of fruit spurs by naked eyes after the epidermal trichome of cucumber fruits have developed to a certain stage, and at the same time, using chemical reagents for fluorescence staining as well as liquid chromatography to determine flavonoids, to assess the effect of gene overexpression in a more comprehensive and accurate way.
This achievement is important in many ways.
First, it brings significant advantages to cucumber itself in terms of nutritional value enhancement. As a common vegetable, the nutritional value of cucumber has always attracted much attention. Through this achievement, the content of flavonoids in cucumber can be increased, and flavonoids have a variety of physiological functions that are beneficial to the human body, such as antioxidant, anti-inflammatory, and prevention of cardiovascular disease. As a result, the nutritional value of cucumber has been strongly enhanced and can provide more health benefits to consumers.
Secondly, in terms of reuse of natural flavonoids, this achievement opens up a completely new way. Flavonoid substances have potential application value in many fields such as medicine, health products and cosmetics. However, the traditional way of obtaining them is often restricted by factors such as limited resources and high extraction costs. By realizing the efficient production of flavonoid substances in the epidermis of cucumber, this research result provides a sustainable method for the large-scale acquisition of natural flavonoid substances, and offers new resources and ideas for the development of related industries.
Finally, this achievement further enhances the ability of cucumber itself to respond to environmental stresses. In the natural environment, cucumber may face a variety of stress factors, such as drought, pests and diseases, and so on. Flavonoids have physiological functions in plants, such as enhancing the antioxidant capacity of plants, regulating the growth and development of plants, and improving the defense capacity of plants against pests and diseases. Therefore, the increased content of flavonoids in the epidermis of cucumber makes the cucumber better able to cope with environmental stresses and improves its ability to survive and grow in complex environments, which is of great practical significance for environmental protection and sustainable development of agriculture.
We hope that we can explore the application of more plants in the field of plant synthetic biology. The research of this project used cucumber as a plant sample and achieved certain results, and we are also thinking whether other plants can increase the production of beneficial metabolites through similar gene regulation mechanisms. We hope that through our joint efforts, we can continuously improve and optimize the research methods of plant synthetic biology.
In addition, we also hope to further study the metabolic pathways in plants. Although this study focuses on the synthesis of flavonoids, there are many other important metabolic pathways in plants. In the future, we can further explore the key genes and regulatory mechanisms in other metabolic pathways, such as alkaloids and terpenoids. Through a deeper understanding of these metabolic pathways, it is possible to develop more biologically active natural products and provide more resources for the fields of medicine, food and agriculture.