Project Description

Epidermal trichome are specialized structures of the above-ground tissues of plants, which have important functions in the plant's own growth and development, in the human chemical industry, and in the pharmaceutical industry. Epidermal trichome are categorized into glandular and non-glandular epidermal trichome according to their secretory capacity. It is often believed that glandular epidermal trichome play a key role in the synthesis and secretion of plant metabolites. Cucumber, as an important vegetable species worldwide, has fruit spines on the fruit epidermis that belong to the epidermal trichome. Fruit spines, as an important cosmetic quality trait, affect the economic value of cucumber to a greater extent in the markets of Asian countries. At the same time, cucumber fruit spines are also important sites for the synthesis of important secondary metabolites. Among the secondary metabolites, flavonoids are important natural phenols. Flavonoids have important physiological functions, in addition to the plant's own protection against ultraviolet radiation and high intensity light, but also beneficial to human treatment of cancer, cardiovascular disease, diabetes, inflammation, obesity and aging and other chronic diseases. However, the utilization of cucumber fruit spines as a bioreactor for the production of flavonoids has not been reported yet. Therefore, this project attempts to utilize cucumber fruit spines as a bioreactor for flavonoids to obtain more flavonoids. Through in-house discussions and discussions with experts related to cucumber epidermal trichome development, we finally selected CsTBH, a fruit spur development gene, and CsPAL, a key gene for regulating the rate-limiting rate of flavonoids, as the regulatory modules in anticipation of flavonoid production in cucumber fruit spurs. The results showed that overexpression of the CsTBH-CsPAL module resulted in a significant increase in flavonoid content in cucumber fruit spines. Successful implementation of the system could provide an important theoretical basis for the development of functional vegetables that are needed by specific populations in the future, as policy permits.

Epidermal trichome are specialized structures formed by the differentiation of epidermal cells in the above-ground parts of plants, which are usually classified into glandular Epidermal trichome (glandular hairs) and non-glandular Epidermal trichome (non-glandular hairs) based on the type of secretion (Singh et al., 2019).Glandular hairs are also known as the “biofactory” of plants because of their ability to synthesize and store plant-specific secondary metabolites, and their structure and synthesized and secreted products are important for plant adaptation to the environment and defense against external stresses (Fahn, 2000; Huchelmann et al., 2017). In addition to this, plant glandular hairs have the ability to synthesize, store and secrete secondary metabolites such as terpenoids, flavonoids, phenylpropenes, methyl ketones, and acyl sugars to name a few (Singh et al., 2019). These metabolites are the material basis of particular goods, medicines as well as nutraceuticals. Thus, plant glandular hairs are equally medicinal and economic (Liu et al., 2019). For example, basil (Ocimum basilicum), lavender (Lavandula spica), peppermint (Mentha × piperita), and thyme (Thymus vulgaris) are known for their essential oils, which are produced in their glandular hairs as the main component (Schilmiller et al., 2008). ). Artemisinin, an effective drug for the treatment of malaria, is produced in the glandular hairs of Artemisia annua (Abdin et al., 2003). Cannabinoids synthesized by cannabis glandular hairs have been found to be effective in preventing neurodegenerative and cardiovascular diseases (Pellati et al., 2018). However, due to the lack of glandular hair structures in the model plant Arabidopsis thaliana, research on glandular hairs has mainly focused on horticultural crops such as tomato, Artemisia annua and cucumber (Feng et al., 2021).

(A-A1, cannabinoid-producing mainly Epidermal trichome on the flower stem; B-B1, artemisinin-producing glands and structural formula for artemisinin, image credit: A-A1 (Tanney et al., 2021) ; B-B1 (de Magalhães et al., 2016))

Cucumber (Cucumis sativus L.) is one of the most widely grown vegetable crops and because of its different flavor and hard texture, they have become favorite worldwide. In 2024, worldwide cucumber production was 185.41 billion kilograms (BKg) and China was leading at 77.3 BKg (FAO, 2024). Cucumbers are full of essential nutrients e.g. iron, calcium, protein, minerals and numerous vitamins (Qing et al., 2022). Moreover, cucumber fruit have antioxidant properties because it contains flavonoids, polyols and polysaccharides, which helps in delaying age, and enhance immunity and mental health. Cucurbitacin B and C vitamins in cucumbers are known to prevent liver infections and stops the growth of abnormal and undifferentiated cells that cause tumors and brain injuries. They are also been used in traditional herbal medications.

Top flowers with thorns are the standard for Asian consumers to buy cucumbers. Therefore, spines (specialized Epidermal trichome on the fruit) are of particular importance. Cucumber fruit spines are multicellular Epidermal trichome and are often used as one of the best models for studying cell division, differentiation, and many other aspects of biology. As also mentioned above, fruit spines belong to Epidermal trichome. In cucumber, the developmental process can be divided into five stages: initiation, first division, glandular hair head differentiation, glandular hair head formation and metabolite synthesis (Dong et al., 2022). It is not clear whether some of the genes regulating epidermal trichome development, such as TBH, are involved in the formation of important secondary metabolites such as flavonoids (Zhang et al., 2021; Dong et al., 2022; Feng et al., 2023).

Flavonoids are secondary metabolites of phenylpropanoid initiation that are widely found in plants, and include flavonoids, flavonoids, anthocyanins, proanthocyanidins, flavonols, flavanediols, and zeaxanthins. Because flavonoids tend to have distinct optical properties, glandular hairs containing such substances can be observed under a fluorescence microscope with distinctive fluorescence. Flavonoids are usually found as water-soluble glycosides in cell vesicles and colored plastids, which can absorb ultraviolet rays and act as “sunscreen” to protect plant cells and enhance their resistance to UV stress, as well as being anti-insect, anti-bacterial, and anti-viral (Saleh et al., 2020). For example, the leaf glandular hairs of Rhodiola rosea are able to synthesize and secrete flavonoid glycosides in response to excessive sunlight, which enhances resistance to intense light radiation (Tattini et al., 2000). Additionally flavonoids are beneficial to human health, with studies suggesting that they can help prevent and treat chronic diseases such as cancer, cardiovascular disease, diabetes, inflammation, obesity and aging (Lin et al.,2017; Putta et al.,2018; Sharma et al.,2018; Kimble et al.,2019).

The core pathway of flavonoid biosynthesis is very conserved and belongs to 1 branch of the phenylalanine analog pathway, which is the model system of plant metabolism (Fig.1). The biosynthesis of flavonoids is mainly controlled by structural genes and regulatory genes (Naing et al., 2018), and the unstable flavonoids are first formed under the catalytic action of enzymes encoded by structural genes, and then through a series of glucosyltransferase (GT), acyltransferase (AT), and methyltransferase (Methyltransferase). The more stable form is catalyzed by glucosyltransferase (GT), acyltransferase (AT), and methyltransferase (MTs). The transcript levels of these structural genes are mainly regulated by the MBW (MYB-bHLH-WD40) transcription complex (Xu et al., 2015). However, it remains unclear whether flavonoids are regulated by HD-ZIP transcription factor (CsTBH belongs to HD-ZIP transcription factor) during epidermal trichome development and product metabolism.

Flavonoids are a widespread class of secondary metabolites that play an important role in helping plants resist environmental stress. The previous researchers also found that the flavonoid content of insect-resistant varieties increased rapidly after being damaged by whiteflies or aphids and was always higher than that of insect-susceptible varieties when they were screened for whitefly- and aphid-resistant cucumber varieties (Xu, Jianpeng, 2016).Wei et al. (2016) had analyzed the volatile chemical compounds qualitatively and quantitatively in 23 different tissues and organs such as roots, stems, leaves, flowers, and fruits in cucumbers and found that the different tissues of mature plants (12 weeks old) had different volatile organic compounds in different tissues. Fengxue et al. (2022) detected a total of 744 metabolites in cucumber glandular hairs using liquid chromatography mass spectrometry (LC-MS/MS), which were classified into 11 major groups based on their chemical structures, with phenylpropanoids and polyketides having the highest relative contents, followed by alkaloids and their derivatives, nucleosides/nucleotides and their analogs, organic acids and their derivatives, benzene, lipids and lipoid compounds, organic heterocyclic compounds, organic oxygenates, organic nitrogen compounds, and lignin compounds, in order of prevalence. compounds, organic nitrogen compounds and lignin. Although the relative content of lipids and lipids is low, the largest number of species is present. Among the 158 phenylpropanoids and polyketides with the highest relative content, there were 114 flavonoids, of which the most abundant were isoquercitrin, zingiberoside, and delphinidin-3-O-glucoside. The above findings in the Epidermal trichome of cucumber glands indicate that flavonoids are indeed present in the plant. However, how to produce more flavonoids in the Epidermal trichome is one of the difficulties in the current study.

Through the organization of the above literature, it was found that a certain content of flavonoid substances existed in the Epidermal trichome of cucumber glands (Feng Xue, 2022). Meanwhile, the developmental process of cucumber Epidermal trichome and the key genes for its regulation have also been reported (Zhang et al., 2021; Dong et al., 2022). The integrated team members together with experts in cucumber epidermal trichome development discussed that by using biotechnological means, the key synthesizing genes of flavonoids could be expressed in high amounts in specific Epidermal trichome during the critical period of epidermal trichome development, and the expected result would be the detection of high concentrations of flavonoids in epidermal trichome cells of cucumber fruits. Therefore, we utilized CsTBH, a key gene controlling the development of Epidermal trichome, and CsPAL, a key rate-limiting enzyme gene for synthesizing flavonoid substances, were used as the design units, and it was confirmed that CsTBH could activate the expression of CsPAL; on this basis, the co-expression of the above mentioned even two expression elements ensured the production of flavonoid substances by the CsTBH-CsPAL module in the epidermis (Fig.5). Utilizing this design scheme, flavonoid substances can be produced amicably to increase the nutritional value of cucumber. More importantly, using this design principle, it provides a feasible solution for human industrial utilization by modifying the Epidermal trichome of plants as a plant factory for flavonoid substance production.

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