Zanthoxylum bungeanum Maxim. (genus: Zanthoxylum; family Rutaceae) has recently gained significant attention from researchers because of its applications in the pharmaceutical, food, and cosmetic industries (Sun et al., 2019). They are widely distributed in the tropical and subtropical regions, including China, Japan, Korea, India, etc (Sun et al., 2019). To date, over 250 species have been identified globally in this genus, with China harboring at least 45 species (Sun et al., 2019). It contains a variety of health-beneficial bioactive compounds. Numerous studies have focused on the chemical structure and bioactivity of different compounds, including flavonoids, alkaloids, amides, lignans, and coumarins. Among these, alkylamides are known for the pungent flavor and pharmaceutical properties of Z. bungeanum. Twenty-seven alkylamides have been identified and characterized as unsaturated fatty acid amides (Zhao et al., 2013). Alkylamides are formed through conjunction with phenylalanine or valine along with unsaturated fatty acids. These compounds have diverse properties such as antiviral, antifungal, antibacterial, antidiabetic, anti-inflammatory, analgesic, antinociceptive, antioxidant, antithrombotic, antimutagenic, anticancer, antimalarial, antitrypanosomal, psychopharmacological, androgenic, spermatogenic, and insecticidal. They also have valuable immunomodulatory and ethnomedicinal uses and are effective in the treatment of anesthesia, neurodegeneration, and skin disorders (Greger, 1984). Therefore, it is imperative to study the natural origin and production of alkylamides due to their numerous benefits in medicine, food, and cosmetics. To date, most studies have focused on the identification and isolation of these compounds, while very few studies have studied the molecular biology behind their biosynthesis. Deciphering the biosynthesis pathways will not only enhance the understanding of the mechanism of action, but also help to augment their production through metabolic engineering. To achieve this, it is necessary to elucidate the numerous biosynthesis pathways, primary precursors, and genes involved in alkylamide regulation and biosynthesis. Previously, a transcriptome analysis of three varieties of Z. bungeanum was carried out (Wu et al., 2020), and 49 genes were expressed differentially. These genes are speculated to have roles in the alkylamide biosynthesis pathways. Therefore, the present study aims to determine the role of the ZbFAD2 and ZbFAD3 genes in the alkylamide biosynthesis pathway.

Z. bungeanum seeds contain over 90% unsaturated fatty acids (Sun et al., 2016). These are essential fatty acids as they cannot be synthesized by the human body (Stoutjesdijk et al., 2000). Their α- linolenic acid and linoleic acid contents are as high as ~70%. Fatty acid desaturases are extremely important in alkylamide biosynthesis. They desaturate most glycerolipids in plants, either membrane-bound or soluble and are present in the endoplasmic reticulum (ER) and chloroplasts (Bryant and Mezine, 1999). FAD2 and FAD3 mostly desaturate extra-chloroplastic lipids that are found as integral ER membrane-bound proteins (Los and Murata, 1998). In the ER, FAD2 destaurates oleic acid (18:1) into linoleic acid (18:2), while FAD3 destaurates linoleic acid into γ-linolenic acid (C18:3, n6) (Bhunia et al., 2016). During fatty acid desaturation, stearoyl-ACP desaturase first converts stearic acid into oleic acid in advanced plants. Then, the fatty acid dehydrogenase converts the unsaturated bonds into an acyl chain at particular positions (Los and Murata, 1998; Bhunia et al., 2016). The sensation of pungency related to Z. bungeanum is conferred by aliphatic alkylamides with unsaturated fatty acid chains and N-terminal isobutyl structures. Numerous studies on alkylamides have demonstrated the taste and bioactivities of Z. bungeanum (Caterina et al., 1997; Story et al., 2003; Bhunia et al., 2016; Zhang et al., 2019). Hydroxy-ϵ-sanshool, hydroxy-α-sanshool, hydroxy-β-sanshool, hydroxy-γ-sanshool, bungeanool, and isobungeanool are the main sources of alkylamides in Z. bungeanum. However, hydroxy-sanshool has been reported to affect the intensity of pungency in Z. bungeanum (Jordt et al., 2004). The molecular mechanisms of some alkylamide bioactivities have been deciphered, e.g., they display anticancer properties by disrupting the mitochondrion-dependent apoptotic pathway, thereby inhibiting cell survival (Jordt et al., 2004; Sugai et al., 2005). These compounds also improve glucose and lipid metabolism by modulating the A δ mechanosensory nociceptor activity and the pathway of the adenosine monophosphate-activated protein kinase, thereby affecting diabetes (Tsunozaki et al., 2013). Hydroxy-sanshool has been shown to decrease the occurrence of skin wrinkles (Kawai et al., 2013). It can also improve memory and learning by modulating the brain’s neuronal terminals (Sugai et al., 2005; Riera et al., 2009).

Alkylamides are considered a combination of unsaturated fatty acids, valine, or phenylalanine. However, the alkylamide biosynthesis pathway, critical precursors, and their associated genes in Z. bungeanum are yet to be identified. Although alkylamide compounds have been proven to be the key components of Z. bungeanum, their biosynthesis pathways remain largely unknown. Hydroxyl-α-sanshool is considered an important alkylamide variant. Upon examining the transcriptomes of the three Z. bungeanum varieties with significantly different hydroxyl-α-sanshool levels, it was found that several DEGs were associated with the unsaturated fatty acid, valine, and leucine biosynthesis tended to affect alkylamide synthesis (Rong et al., 2016). Plants are the only organisms capable of de novo fatty acid synthesis in the plastids, which include acetyl-CoA, palmitic acid (16:0), stearic acid (18:0), and oleic acid (18:1△⁹). These are then further desaturated to form linoleic acid (18:2△^9,12) and linolenic acid (18:3△^9,12,15) via the prokaryotic pathway. However, the eukaryotic pathway occurs via the ER (Koo et al., 2007). In the ER, FAD2 catalyzes the first step of the polyunsaturated fatty acid biosynthesis, converting oleic acid to linoleic acid and linolenic acid by incorporating a double bond between carbon 12 and 13 at the sn-1 and sn-2 sites in phosphatidylcholine. Additionally, omega-6 FAD2 and omega-3 FAD (FAD3) convert oleic acid (C18:1Δ9) to linoleic acid, linoleic acid, and α-linolenic acid (C18:3Δ^9,12,15). FAD3, a linoleate desaturase, regulates the amount of C18:3 in the seed oil by converting the phosphatidylcholine-bound linoleate to linoleate in the ER. However, there are limited studies on the relationship of biosynthetic pathways between polyunsaturated FADs and alkylamides in Z. bungeanum. Furthermore, the whole genome study of Z. bungeanum has shown that ZbFAD2 and ZbFAD3 are crucial genes in sanshool biosynthesis (You et al., 2015).

The present study aimed to functionally validate the ZbFAD2 and ZbFAD3 genes, as well as evaluate the correlation between unsaturated fatty acids and alkylamide compounds. The findings will provide a theoretical basis for the research on the cannabinoids biosynthesis pathway in Z. bungeanum. Furthermore, the molecular research on this pathway in Z. bungeanum provides technical support for the accumulation and directional regulation of the compounds in Z. bungeanum, which has both theoretical significance and economic value.

In recent years, Z. bungeanum has increasingly been proven to be a valuable source of bioactive compounds, which endowed it with several human health beneficial properties. Along with the pharmaceutical industry, both the cosmetics and food industries have also benefited. In this regard, numerous classes of compounds, including alkylamides, from Z. bungeanum have been explored and characterized. Alkylamides not only have an extensive range of bioactivities and applications, but also impart a distinct tingling sensation onto mucosal surfaces post application (Tsunozaki et al., 2013). Sanshools are valuable natural alkylamides that cause tingling and paresthesia when applied to the skin or tongue. These belong to a family of polyunsaturated fatty acid amides and constitute an essential flavor and medicinal ingredient in Z. bungeanum, which can be used as metabolic markers to evaluate Z. bungeanum quality. It is important to analyze different plant varieties, their gene expression, and the genetic differences in the Z. bungeanum biosynthetic pathways (Bhatt et al., 2017) for screening and improving its germplasm. Most studies have focused on their biological activities, extraction, and separation, but fewer have studied their secondary metabolism pathway and the synthetic regulation of sanshools. Therefore, the sanshool biosynthesis pathways and the associated active players involved in it still remain obscure (Cortez-Espinosa et al., 2011; Ren et al., 2017; Wu et al., 2020). The transcriptome sequencing of Z. bungeanum helped identify 19 DEGs related to unsaturated fatty acid, isoleucine, leucine, and valine biosynthesis. Keeping this in mind, our study results confirmed the importance of the ZbFAD3 and ZbFAD2 proteins in the alkylamide biosynthesis pathway.

This is the first time that Arabidopsis thaliana has been used to express the ZbFAD2 and ZbFAD3 proteins. Although FAD2 and FAD3, which were previously thought to transform oleic acid into linoleic acid, had been studied in different plants, no reports were found on the cloning and subsequent characterization of these desaturases from Z. bungeanum. The current work is the first report on the isolation of FAD2 and FAD3 from this plant. We performed standard bioinformatics analyses to determine their role in the alkylamide biosynthesis pathways. Through phylogenetic analysis of both desaturases, and the results obtained from gene function verification, we found that these genes were closely associated with PtFAD2 and PeFAD2. This will be helpful in managing the demand pressure of alkylamides in the medicine, cosmetics, and food industry (Xue et al., 2017; Gazave et al., 2020; Feng et al., 2021).

Both desaturases (FAD2 and FAD3) are part of a larger family (FAD) of the membrane FADS-like superfamily. Five transmembrane helices, composed of a random coil, beta-turn, and alpha helix, were found in the secondary structures of FAD2 and FAD3. According to the transcriptomic study of Z. bungeanum, FAD5, FAD6, FAD7, and FAD8 were also involved in the biosynthesis of fatty acids. FAD2 and FAD6 converted oleic acid into linoleic acid via desaturation, whereas FAD3, FAD7, and FAD8 converted linoleic acid into α-linolenic acid through desaturation. This highlights the potential of using Z. bungeanum for synthesizing linoleic acid (Chen et al., 2014; Busch et al., 2020). Increasing linoleic acid content in plants is mainly achieved through their trait-directed breeding. The expression of FAD2 in Brassica napus and Brassica juncea increased their oleic acid content to 89% and 73%, respectively (Horiguchi et al., 2000). FAD3 gene has been successfully cloned in Arabidopsis (Arondel et al., 1992), tobacco (Shimada et al., 2000; Orlova et al., 2003), wheat (Horiguchi et al., 2000), flax (Vrinten et al., 2005), rapeseed and other plants. It has been found that inhibiting the expression of FAD3 in Arabidopsis thaliana or other plants, that is, inactivating and mutating it, will cause the content of 18:3 in the transgenic lines to decrease and the content of 18:2 to increase (Rahman et al., 1996). On the contrary, overexpression of FAD3 gene will increase the 18:3 content in transgenic lines (Shimada et al., 2000; Orlova et al., 2003; Shah and Xin, 1997).

Ectopic expression of FAD2 and FAD3 genes in the Arachis hypogaea, Sesamum indicum, Helianthusannuus, etc different plants have shown that these genes could convert oleic acid into linoleic acid and generate hexadecadienoic acid (16:2 Δ^9,12), using palmitoleic acid (16:1Δ⁹) as a catalyzed substrate. This may be attributed to some specific substrate preference or varied transmembrane topologies found in the FAD2s and FAD3s (Huang et al., 2010; Fan et al., 2019).

Three Z. bungeanum varieties were subjected to transcriptomic analysis in a previous study (Wu et al., 2020). Based on the obtained data, we found that all alkylamide biosynthesis-related genes were expressed in the three varieties, with varying degrees of expression. However, some FAD genes exhibited differences in transcript levels, which were consistent with previous findings. Upon performing a homologous sequence comparison based on the cDNA sequences of these FAD genes, we identified them as belonging to the unsaturated fatty acid dehydrogenase family and subsequently designated them as ZbFAD2 and ZbFAD3. The lengths of the ORFs of these enzymes were determined to be 1,167 bp and 1,152 bp, respectively. By comparing the expression pattern and linoleic content, we found that the expression levels of ZbFAD2 and ZbFAD3 were positively correlated with the linoleic acid concentration of Z. bungeanum. The findings demonstrate that ZbFAD2 and ZbFAD3 significantly affect the accumulation and regulation of polyunsaturated fatty acids (PUFAs), by converting oleic acid to linoleic acid at different growth stages of Z. bungeanum.

Based on the results of bioinformatics analysis and prokaryotic expression, we found that ZbFAD2 and ZbFAD3 encoded ER-located-like FAD2 and FAD3 enzymes. The functional characterization of desaturases in A. thaliana tested the ability of ZbFAD2 and ZbFAD3 proteins in catalyzing linoleic acid synthesis. Our study provides proteomic data for the molecular regulation and coordination in Z. bungeanum with high linoleic acid content. Further characterization of ZbFAD2 and ZbFAD3 and their functional relationship may provide more information on the mechanisms controlling the fatty acid composition of Z. bungeanum, which may help in trait improvement via both genetic engineering and breeding.

The molecular weight of both purified proteins was very close to the bioinformatically-predicted value. Our findings showed that ZbFAD2 reacted with oleic acid (C18:1△⁹) to produce linoleic acid (C18:2△^9,12), while ZbFAD3 potentially reacted with linoleic acid to produce α-linolenic acid (C18:3△^9,12,15) ( Figure 4 ). The homologous sequence analysis revealed that ZbFAD2 and ZbFAD3 could catalyze the conversion from oleic acid to linoleic acid, and the conversion from linoleic acid to α-linolenic acid, respectively. Therefore, both unsaturated fatty acid dehydrogenases function by introducing a second and third double bond to the fatty acid chain.

It was observed that the linoleic acid and oleic acid contents in the ZbFAD2-A. thaliana transgenic seeds were higher and lower than in the WT, respectively. However, the levels of α-linolenic acid and linoleic acid in the ZbFAD3-A. thaliana transgenic seeds were higher and lower than in the WT, respectively. Our results indicated that the recombinant ZbFAD2 and ZbFAD3 proteins functioned as desaturase enzymes and could catalyze unsaturated fatty acid dehydrogenation; which agreed with the prokaryotic expression analysis results, thereby confirming their roles in the unsaturated fatty acid biosynthesis pathway.

At present, both FAD2 and FAD3 genes have been successfully cloned in perilla, tobacco, Arabidopsis, wheat, flax, etc. The content of 18:3 in rice bran oil and roots increased significantly after the soybean FAD2 and FAD3 genes were transformed into rice, with the α-linolenic acid content in rice bran oil increasing significantly. These results show that overexpression of FAD2 and FAD3 genes can increase the α-linolenic acid content in plants. Therefore, we speculate that both ZbFAD2 and ZbFAD3 genes have an important role in the alkylamide biosynthesis pathway.

To sum up, the prokaryotic and eukaryotic expression vectors of ZbFAD2 and ZbFAD3 genes were constructed, and their functions were evaluated via substrate reactions. Furthermore, expressing them in A. thaliana and N. benthamiana could identify their functional roles in the alkylamide biosynthesis pathway. The difference in the levels of unsaturated fatty acids and hydroxy-α-sanshool indicated that the two target genes were involved in the alkylamide biosynthesis pathway, and fatty acid synthesis was correlated with alkylamide synthesis. Our transient assays on N. benthamiana leaves and stable transformation on A. thaliana demonstrated that the expression of ZbFAD2 and ZbFAD3 genes induced the expression of some key genes for the biosynthesis of hydroxy-α-sanshool. It was observed that Z. bungeanum possessed an updated pathway for hydroxyl-α-sanshool biosynthesis ( Figure S4 ). Therefore, ZbFAD2 and ZbFAD3 are the crucial enzymes for regulating the alkylamide biosynthesis pathway in Z. bungeanum. These would lay the groundwork for further research of the sanshools in the plants. Nevertheless, future studies should investigate the relationship between the biosynthesis of fatty acids and alkylamide compounds in Z. bungeanum to gain further insights into the alkylamide biosynthesis pathway.

The original contributions presented in the study are included in the article/ Supplementary Materials . Further inquiries can be directed to the corresponding author.

JZ performed the statistical analysis and drafted the manuscript. ZW and NH participated in the statistical analysis and the writing of the manuscript. DW conceived the research, participated in the research design and coordination, and provided suggestions on the writing of the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by the National Natural Science Foundation of China (31872706), and the National Key Research and Development Program of China (2019YFD1000603).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.