Global warming and ecological environment changes have worsened the living conditions of food and horticultural crops. Affected by various biotic and abiotic stresses, the yield and quality are compromised, posing a threat to human food security [1-2]（Picture1 / Picture2). While what is heartbreaking is that, while production capacity is increasing, the issue of global food, fruit, and vegetable waste is shockingly prominent——According to data from the United Nations Food and Agriculture Organization, every year about one-third of the world's food (approximately 1.3 billion tons) and nearly half of fruits and vegetables are lost or wasted. This food, which could have satisfied the needs of hundreds of millions of people, being discarded for nothing in various links of the supply chain or at the consumer end [3]（Picture 3）. Faced with such severe resource depletion, traditional prevention and control methods still rely on the extensive spraying of pesticides [4]. This chemical control method not only affects crop quality and increases production costs, but also violates the concept of green agricultural development [5].
Therefore, it is increasingly urgent to explore more efficient and rapid prevention and control pathways. Focusing on plants themselves and studying how to actively resist adverse factors can not only enhance production capacity but also reduce waste caused by poor crop quality and poor storability at the source. This is undoubtedly a more efficient solution [6].
About 450 million years ago, aquatic plants began to migrate onto land. During this important evolutionary process, the important reason for the continued survival of terrestrial plants is the presence of a cuticle on their surface [7, 8, 9].This hydrophobic outer layer enables plants to survive in terrestrial environments by retaining water. The cuticle is composed of cutin and epidermal wax produced by epidermal cells, and the outermost layer of the plant's cuticle is mainly composed of deposited wax crystals [8]（Picture 4/ Picture 5）.
Plant epidermal wax, as an important lipid-soluble secondary metabolite, not only reducing non-stomatal water loss from the plant epidermis, enhances drought resistance, and improves storability, but also resists temperature stress and prevents UV damage [10].
More importantly, it serves as a crucial physical barrier for plants to resist pathogen infection, exerting both structural and chemical disease resistance [11-12]. Waxiness not only affects the mechanical strength of plant epidermis but also hinders the attachment of pathogens, effectively reducing pathogen infection. Certain components of waxiness can inhibit or stimulate spore germination [13].
Most plant epidermis in nature contains a layer of hydrophobic lipid compounds, known as waxes, which protect plants from harsh external environments and pathogenic bacteria [14]. Waxy substances are composed of esters with ultra-long chain aliphatic groups with carbon chain lengths greater than C20, triterpenoids, and secondary metabolites such as alcohols and flavonoids [15]. Waxy components can be divided into long-chain compounds and cyclic compounds. Among them, long-chain compounds are mostly derivatives of very long-chain fatty acids (VLCFA), including alcohols, alkanes, esters, aldehydes, ketones, etc. Cyclic compounds include aromatic compounds and alicyclic compounds [16]. Waxiness plays a crucial and diverse role for plants, helping them reduce non-stomatal water loss, resist ultraviolet radiation (UV), defend against pathogens and pests, and enhance surface mechanical strength [17], Wax also plays an important role in our lives. Many plant epidermal waxes can be extracted and used as natural raw materials in various industries. For example, they can be made into edible fruit wax and sprayed onto the surface of fruits and vegetables for preservation and coating [18], As an important ingredient in many cosmetics, pharmaceuticals, and daily necessities [19], it can also be extracted and used as a polishing agent and protective agent in the automotive industry [20]. However, plants grown in their natural state have very low wax content, and industrial production requires a large planting scale with high costs.
The synthesis, transportation, and regulation of plant wax are highly complex processes involving the synergistic action of multiple genes and transcription factors. Alkanes, as the main component of most plant waxes, account for 50%-80% of the total wax content [21-22]. Therefore, we can explore the key pathway controlling the synthesis of paraffin waxes and modify it to increase wax content [23-24].The synthesis of alkanes in Arabidopsis thaliana has been extensively studied, and the alkanes synthesis gene ECERIFERUM1 (CER1) has been identified and investigated. In the Arabidopsis thaliana cer1 mutant, the content of alkanes and their derivatives decreases significantly, while the content of aldehydes increases [25-26].Therefore, it is indicated that there is a positive correlation between the expression level of CER1 and wax content. Some studies have reported that the CER1 pathway is likely to be influenced by light exposure, but the specific internal mechanism has not been elucidated [27].
We designed an experiment using Nicotiana benthamiana to first verify whether there is a pathway in plants where the light-synthesized gene HY5 combines with CER1 to jointly control wax synthesis. Then, we altered external light conditions to modify this pathway, enhancing the binding ability of HY5 and CER1 promoters, thereby increasing the expression level of the alkane synthesis gene CER1. This led to a significant increase in wax content in plants, improving their inherent resistance and providing abundant raw materials for the industrial sector.
Plant epidermal wax synthesis pathway [14]
Comparison of drought resistance in rice varieties with
different wax content [15]
Our research is based on synthetic biology principles to develop a high-efficiency and high-yield wax synthesis system. Using Nicotiana benthamiana as a vector, we enhance the wax content in tobacco plants by modifying the wax synthesis and metabolic pathways within the plant. On the one hand, this improves the plant's own stress resistance and creates new high-wax germplasm resources. On the other hand, it provides a low-cost, green, and sustainable industrial raw material. Specifically, we clarified the relationship between the light regulatory element HY5 and the wax synthesis-related gene CER1 in plants, and discovered the existence of a HY5-CER1 wax synthesis regulatory pathway in plants. By continuously adjusting and optimizing light conditions, we enhanced the binding ability of HY5 and CER1, maximizing the expression of CER1, and thereby improving the level of wax synthesis in plants.
Overall, our research utilizes synthetic biology principles to transform Nicotiana benthamiana into an efficient "green factory", and by revealing a new mechanism of light-regulated wax synthesis, we have achieved a perfect combination of scientific discovery (HY5-CER1 pathway) and technological application (low-cost production of industrial waxes).This research is not only of great significance to agricultural production (stress-resistant crop breeding), but also provides humanity with a new sustainable and low-cost approach to producing industrial raw materials, possessing immense economic potential and environmental value.
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