1 Introduction

Isoflavones are mainly found in leguminous plants (mainly in soybeans) and are secondary metabolites. They play an important role in plant defense mechanisms, can enhance plant disease resistance and stress resistance, and are very important for soybean production and development (Veremeichik et al., 2020; Sohn et al., 2021). In addition to their role in plants, isoflavones are also important in the field of human health due to their estrogenic activity and antioxidant properties. Studies have shown that the intake of isoflavones is closely related to a reduced risk of hormone-dependent cancers, osteoporosis, menopausal syndrome, and cardiovascular disease. Therefore, isoflavones are also important bioactive components in the human diet (Jung et al., 2000; Sohn et al., 2021).

The biosynthesis of soy isoflavones is a complex process branching from the phenylpropanoid metabolic pathway, involving multiple key enzymes. Among them, Isoflavone Synthase (IFS) catalyzes the initial step of the biosynthesis pathway, while Chalcone Synthase (CHS), Chalcone Reductase (CHR), Chalcone Isomerase (CHI) and other enzymes jointly participate in the regulation of this pathway and form metabolic complexes anchored to the endoplasmic reticulum (Jung et al., 2000; Dastmalchi et al., 2016). In addition, the transcriptional regulation of this pathway is finely regulated by multiple transcription factors, including GmMYB29 of the R2R3-MYB family and GmZFP7 of the C2H2 zinc finger transcription factor, which affect the biosynthesis of isoflavones by regulating the expression levels of key enzyme genes (Chu et al., 2017; Feng et al., 2022). At present, metabolic engineering has become an important strategy for increasing the content of soy isoflavones. Relevant studies have shown that by regulating key genes in the biosynthetic pathway, soybean disease resistance can be enhanced and its nutritional value can be improved (Yu et al., 2003; Yu and McGonigle, 2005).

This study will systematically review the latest research progress in soybean isoflavone biosynthesis from the perspective of metabolic regulation, focusing on the key enzyme functions in the synthesis pathway, transcriptional regulation mechanisms, and the development of metabolic engineering technology. It is hoped that it will provide theoretical support for molecular breeding and functional food research and development to increase the content of soybean isoflavones, and analyze the potential application value of isoflavones in soybean agricultural production and human health.

2. Biochemical Pathways of Isoflavone Biosynthesis

2.1 Key precursors and initial steps for isoflavone formation

The biosynthesis of soy isoflavones begins with the phenylpropanoid metabolic pathway, which provides the necessary precursor molecules for the synthesis of flavonoids. The initial step of biosynthesis is catalyzed by phenylalanine ammonia lyase (PAL), which converts phenylalanine from ammonia to cinnamic acid. Subsequently, under the catalysis of cinnamate 4-hydroxylase (C4H), cinnamic acid was further hydroxylated to form p-coumaric acid (Dastmalchi et al., 2016). On this basis, Chalcone Synthase (CHS) catalyzes the condensation reaction of multiple precursor molecules to form naringenin chalcone, which is considered a key rate limiting step in the synthesis of flavonoids and marks the official start of flavonoid biosynthesis (Imaizumi et al., 2020).

2.2 Functions of chalcone synthase (CHS) and isoflavone synthase (IFS)

Chalcone Synthase (CHS) is a key rate limiting enzyme in the biosynthesis pathway of isoflavones, catalyzing the condensation of p-coumaroyl-CoA and malonyl CoA to produce naringenin chalcone (Imaizumi et al., 2020). CHS not only serves as the core catalytic enzyme of this pathway, but also forms metabolic complexes with other flavonoid biosynthetic enzymes, known as isoflavone metabolome. Its function is to improve metabolic efficiency through substrate channel effects and optimize the synthesis rate of isoflavones (Dastmalchi et al., 2016; Waki et al., 2016). Isoflavone Synthase (IFS) is a cytochrome P450 monooxygenase that catalyzes the conversion of naringenin to 2-hydroxyisoflavones, which then undergo spontaneous dehydration to produce daidzein and genistein (Waki et al., 2016; Mameda et al., 2018). The activity of IFS largely determines the flow of isoflavone synthesis, therefore, its expression regulation is crucial for controlling the accumulation of isoflavone compounds.

2.3 Intermediate pathways involved in the synthesis of daidzein and genistein

The biosynthesis of daidzein and genistein involves multiple intermediate metabolites, among which naringenin chalcone is a key precursor that undergoes isomerization under the catalytic action of chalcone isomerase (CHI) to naringenin (Waki et al., 2016). Subsequently, naringin was catalyzed by IFS to undergo hydroxylation, forming 2-hydroxyisoflavones, which were then dehydrated to produce daidzein and genistein (Waki et al., 2016; Mameda et al., 2018). This metabolic pathway is co regulated by multiple enzymes to ensure stable and efficient metabolic flux (Dastmalchi et al., 2016). In addition, Chalcone Reductase (CHR) plays an important role in the biosynthesis of 5-deoxyisoflavones (such as daidzein), catalyzing the reduction of coumaroyl CoA to produce 6 '- deoxychalcone. This reaction provides CHS specific substrates and further promotes the branching metabolic pathways of isoflavone biosynthesis (Mameda et al., 2018).

3 Regulatory Genes of Isoflavone Biosynthesis

3.1 Transcription factors regulating the isoflavone biosynthesis pathway

Transcription factors play a vital role in the transcriptional regulatory network of soybean isoflavone biosynthesis. GmMYB29, a member of the R2R3-MYB family, is one of the core regulatory factors of isoflavone biosynthesis. It can directly activate the promoters of key enzyme genes (such as IFS2 (isoflavone synthase 2) and CHS8 (chalcone synthase 8)). In their 2017 study, Chu et al. found that overexpression of GmMYB29 significantly increased the accumulation of isoflavones, while gene silencing led to a decrease in its synthesis level. The C2H2-type zinc finger protein GmZFP7 is also a key regulator of isoflavone metabolic flux. By upregulating the expression of GmIFS2 and GmF3H1 (flavanone 3 β-hydroxylase 1), GmZFP7 can promote the accumulation of flavonoids and direct the metabolic flow of flavonoids to the flavonoid biosynthesis pathway (Feng et al., 2022) (Figure 1). In recent years, several new MYB transcription factors (such as GmMYB102, GmMYB280 and GmMYB502) have been identified as potential regulatory factors. Overexpression of these transcription factors can significantly enhance the synthesis of isoflavones in transgenic soybean hairy roots, further demonstrating the importance of the MYB transcription factor family in regulating isoflavone biosynthesis (Sarkar et al., 2019).

3.2 Co-regulation with other secondary metabolic pathways

The biosynthesis of soybean isoflavones is closely related to other secondary metabolic pathways, especially the phenylpropanoid pathway. Phenylpropanoid pathway genes are activated by transcription factors such as maize C1 and R. Yu et al. (2003) found that these transcription factors can increase isoflavone levels by regulating the flux of the pathway. Feng et al. found in 2022 that GmZFP7 can exert its effects by affecting the phenylpropanoid pathway, increase isoflavone content, and regulate stress resistance mechanisms (Figure 1). In addition, the diurnal regulation of isoflavone and soybean saponin biosynthesis genes shows a coordinated expression pattern that is consistent with the metabolic needs of plants throughout the day (Matsuda et al., 2020). This co-regulation ensures that metabolic flux is effectively used for the production of isoflavones and other essential secondary metabolites.

3.3 Genetic modification to increase isoflavone content

Genetic modification technology is an important strategy to increase the content of soybean isoflavones, in which the regulation of transcription factors plays an important role in this technology. Studies by Chu et al. (2017) and Feng et al. (2022) both showed that overexpression of GmMYB29 and GmZFP7 can significantly promote gene expression in the isoflavone synthesis pathway, thereby increasing its accumulation level. In addition to directly regulating transcription factors, metabolic engineering strategies are also widely used. Combined inhibition of flavanone 3-hydroxylase (F3H) can effectively block the anthocyanin synthesis branch in the phenylpropanoid metabolic pathway, so that the metabolic flux can be more biased towards the isoflavone synthesis pathway. In a study in 2003, Yu et al. found that when genetic modification technology was co-expressed with transcription factors such as C1 and R that regulate anthocyanin synthesis, the accumulation level of isoflavones would be significantly increased. In a study in 2019, Gupta et al. found that increased cytosine methylation in the coding regions of IFS1 and IFS2 genes was positively correlated with enhanced isoflavone biosynthesis, which provided a new molecular regulation pathway for genetic improvement.

4 Enzymatic Mechanism of Isoflavone Metabolism

4.1 Isoflavone synthase and related enzymes

Isoflavone synthase (IFS) is a key catalytic enzyme in the biosynthesis pathway of soybean isoflavones. Its function is to catalyze the specific conversion of flavonoids to isoflavones and open the synthesis pathway of isoflavone compounds. As a type of cytochrome P450 monooxygenase, IFS is a member of the soybean CYP93C subfamily, and its activity determines the metabolic flux of isoflavone synthesis (Jung et al., 2000; Waki ​​et al., 2016). IFS does not act alone, but forms a metabolic complex (metabolon) with other key enzymes in the phenylpropanoid metabolic pathway, including chalcone synthase (CHS) and chalcone isomerase (CHI). The subcellular localization of IFS and its related enzymes is also important in metabolic regulation. These enzymes are anchored on the endoplasmic reticulum (ER) membrane to form a stable enzyme complex to optimize substrate transport and metabolite synthesis efficiency. This spatial organization structure not only helps to enhance the coordination of metabolic pathways, but also avoids competition from bypass metabolism and ensures the robustness of isoflavone synthesis (Dastmalchi et al., 2016; Waki ​​et al., 2016).

4.2 Enzymatic specificity and reaction kinetics

In the biosynthesis of isoflavones, the substrate specificity of key enzymes determines the synthesis efficiency and product distribution of specific isoflavones (genistein, soy flavonoids, etc.). Isoflavone synthase (IFS) shows high selectivity for its flavanone substrates, and its catalytic activity is not only regulated by its own enzyme structure, but also by the interaction with other metabolic enzymes. Studies by Dastmalchi et al. (2016) and Waki ​​et al. (2016) showed that IFS forms a functional complex with chalcone synthase (CHS) or chalcone isomerase (CHI) to regulate the catalytic efficiency and substrate flow direction of the isoflavone synthesis pathway. The formed complex achieves efficient transfer of metabolic intermediates between enzymes through tightly coupled enzyme-enzyme interactions in space, avoiding diffusion losses. This organizational pattern can significantly improve the overall flux of the synthesis pathway and reduce the possibility of by-product generation.

4.3 Research progress on enzyme engineering for isoflavone production

In recent years, the main research direction of metabolic engineering is to achieve precise regulation of isoflavone metabolic flow by regulating the expression of key enzymes and transcription factors involved in biosynthesis. Studies by Chu et al. (2017) and Feng et al. (2022) both found that overexpression of transcription factors GmMYB29 and GmZFP7 can significantly enhance the expression of IFS and its related enzymes, and improve the overall synthesis efficiency of isoflavones. In order to further optimize the metabolic flow, Yu et al. (2003) also adopted a competitive pathway inhibition strategy (such as combined inhibition of key enzymes in the anthocyanin synthesis pathway). In their study, they effectively reduced the transfer of precursor compounds to anthocyanin metabolites, making more substrates available for the biosynthesis of isoflavones, thereby significantly improving their accumulation level.

5 Impact of Environmental Factors on Isoflavone Levels

5.1 Light and temperature effects on isoflavone accumulation

The accumulation level of isoflavones in soybean seeds is largely influenced by environmental factors, among which the effects of light intensity and temperature conditions are particularly significant. Caldwell et al. (2005) found that increasing temperature can inhibit the synthesis of isoflavones during seed development. When the seed development temperature increased from 18 ° C to 23 ° C, the total content of isoflavones decreased by 65%, and further increased to 28 ° C, the decrease in content was as high as 90%. Chennupati et al. (2012) found that during the late stage of soybean reproduction (R5-R8 stage), when subjected to high temperature stress, the concentration of seed isoflavones decreased by 46-86%. These data indicate that high temperatures not only inhibit the activity of key biosynthetic enzymes, but may also affect transcriptional regulatory networks, leading to a significant decrease in isoflavone metabolic flux. At the same time, lighting conditions are also one of the important factors affecting the accumulation of isoflavones. Barion et al. (2021) found that soybean plants exposed to morning light (east facing) exhibited higher total cotyledon isoflavone concentration (TCIC) compared to those exposed to afternoon light (west facing) (Figure 2). This phenomenon may be related to the role of light signals in regulating gene expression related to the phenylpropanoid pathway. Morning light may be more conducive to activating the expression of isoflavone synthesis related enzymes, thereby increasing metabolic flux.

5.2 Influence of soil nutrients and water availability

Soil moisture has an important influence on the synthesis of isoflavones in soybean seeds. In their 2005 study, Lozovaya et al. found that high soil moisture content is conducive to the accumulation of isoflavones, while water stress significantly inhibits their synthesis. Gutierrez Gonzalez et al. (2010) found that sufficient water can help isoflavones maintain a high metabolic activity and promote the expression of enzymes related to their biosynthesis. The mineral nutrient content of the soil has little effect on the synthesis of isoflavones. The field experiment of Kurosaki et al. (2023) showed that conventional fertilization measures such as potassium (K), nitrogen (N), phosphorus (P), and molybdenum (Mo) had no significant effect on the isoflavone content of soybean seeds. These research results can show that in the process of isoflavone accumulation, water has a more important regulatory role than mineral nutrition. Therefore, in the cultivation and management of soybeans, optimizing water management will be more effective than a simple fertilization strategy.

5.3 Stress-induced isoflavone production in soybean

The accumulation of soy isoflavones is affected by various environmental stresses, including drought, water stress, and mechanical damage. Caldwell et al. found in 2005 that drought stress can significantly affect isoflavone synthesis, but in high carbon dioxide (CO₂) concentration environments, this inhibitory effect may be alleviated, restoring isoflavone levels to control levels under normal growth conditions. This phenomenon may be related to the regulatory effect of CO₂ on carbon metabolism, which in turn affects the metabolic flow of the phenylpropane pathway. Mechanical damage can also promote the accumulation of isoflavones. In 2021, Barion et al. found that leaf damage activates secondary metabolic pathways and increases the synthesis rate of isoflavones, especially under sufficient light conditions, where this effect is more significant. This phenomenon suggests that isoflavones may play an important role in soybean defense responses to help plants cope with biotic and abiotic stresses. In addition to the above two factors, the expression of key genes involved in isoflavone synthesis is also affected by water and temperature. For example, chalcone synthase CHS7 and CHS8, as well as isoflavone synthase IFS1 and IFS2, are regulated by different environmental factors (Gutierrez Gonzalez et al., 2010; Chennupati et al., 2012). Among them, IFS2 is the main regulatory factor for the decrease in isoflavone accumulation under drought conditions, and the decrease in its expression level may be a key mechanism leading to limited metabolic flux.

6 The Role of Hormone Regulation in Isoflavone Biosynthesis

6.1 The role of auxin and cytokinin in the activation of the isoflavone pathway

Auxin and cytokinin are two important plant hormones, both of which play an important role in the biosynthesis of soybean isoflavones. Kumar et al. (2021) found that the application of exogenous serotonin and melatonin is related to auxin signaling, which can increase the expression of isoflavone biosynthesis genes (such as chalcone synthase and isoflavone synthase) and increase the isoflavone content in soybean culture. Chu et al. (2017) found that the transcription factor GmMYB29 regulated by hormone signals can activate the promoters of isoflavone synthase 2 (IFS2) and chalcone synthase 8 (CHS8), further enhancing the biosynthesis of isoflavones.

6.2 Crosstalk between hormones and secondary metabolic pathways

The relationship between plant hormones and secondary metabolic pathways is relatively complex, involving multiple regulatory networks. In 2021, Kumar et al. found that plant hormones such as ethylene (ET), jasmonic acid (JA) and abscisic acid (ABA) play an important role in regulating isoflavone biosynthesis. In soybean tissue culture systems, treatment with serotonin and melatonin can affect ethylene biosynthesis and regulate the expression of genes related to isoflavone biosynthesis. These studies all indicate that there is an interaction between plant hormone signaling pathways and secondary metabolic pathways. Plant hormones can also regulate the accumulation of isoflavones through specific transcription factors, such as GmZFP7, which can act as a C2H2 zinc finger protein transcription factor regulated by hormone signals, affect the expression of key enzyme genes in the phenylpropanoid pathway, and promote the synthesis of isoflavones through the redistribution of metabolic flux (Feng et al., 2022).

6.3 Hormone signals during soybean development

Plant hormones play different roles in different stages of soybean growth and development, profoundly affecting the synthesis and accumulation of isoflavones in soybeans. The rhythmic regulation of root isoflavone biosynthesis is affected by the circadian rhythm, which is in turn regulated by plant hormone signals. Matsuda et al. (2020) found that the expression levels of isoflavone biosynthesis-related genes fluctuated significantly between day and night, and the expression level during the day was significantly higher than that at night, indicating that photoperiod signals and hormone pathways are jointly involved in the metabolic regulation of isoflavones. Chen et al. (2017) and Yang (2024) found that miRNA not only affected the transcription level of isoflavone synthesis-related genes, but also indirectly affected the accumulation of isoflavones by targeting hormone signaling pathways.

7 Applications in Agriculture and Nutritional Improvement

7.1 Breeding strategies for high-isoflavone soybean varieties

The breeding strategy for high-isoflavone soybean varieties is a genetic and molecular mechanism for controlling isoflavone biosynthesis, with the goal of breeding soybean varieties with high isoflavone content. One of the most important steps is to identify the key gene GmMYB29, which can regulate isoflavone biosynthesis by activating the IFS2 and CHS8 gene promoters. The study by Chu et al. in 2017 showed that overexpression of GmMYB29 in soybean hairy roots increased isoflavone content, while RNAi-mediated silencing led to reduced isoflavone levels. In addition, the expression of isoflavone biosynthesis genes can also be affected by the environment. For example, soybean varieties such as Hefeng 25 and Sfera have increased isoflavone content under adverse climatic conditions (Veremeichik et al., 2020). These research results are conducive to the selection and genetic manipulation of soybean genotypes to improve isoflavone content.

7.2 Role of isoflavones in plant defense mechanisms

Isoflavones can act as phytoalexins in leguminous plants such as soybeans, compounds synthesized in response to pathogen attacks, thereby enhancing plant disease resistance and playing an important role in plant defense mechanisms. Soybean roots can secrete isoflavones such as daidzein and genistein, which can affect rhizosphere interactions, including inducing symbiosis with rhizobia (Sugiyama et al., 2017). Under stress conditions (such as the application of methyl jasmonic acid), the expression of isoflavone biosynthesis genes increases, which can further enhance their role in plant defense. Jeong et al. found in 2018 that methyl jasmonic acid treatment can significantly upregulate genes involved in the isoflavone biosynthesis pathway, increase isoflavone production, and enhance plant defense capabilities.

7.3 Isoflavones as functional nutraceuticals

Isoflavones can promote human health and are valuable functional nutrients. Sohn et al. (2021) demonstrated that isoflavones can reduce the incidence of hormone-related cancers, osteoporosis, menopausal symptoms, and cardiovascular diseases (Figure 3). Metabolic engineering of isoflavone biosynthesis has important agronomic and nutritional significance for both leguminous and non-leguminous crops. In Arabidopsis, the expression of soybean isoflavone synthase can produce the isoflavone genistein, indicating that it can increase the dietary isoflavone content of various crops (Jung et al., 2000). Yuk et al. (2016) and Kumar et al. (2021) treated soybeans with ethylene and other signaling molecules (such as serotonin and melatonin) to regulate the accumulation of isoflavones in soybeans. Advances in metabolic engineering and regulation of isoflavone biosynthesis pave the way for the development of healthy crops.

8 Biotechnological Advances in Isoflavone Engineering

8.1 Genetic engineering approaches for isoflavone biosynthesis

Genetic engineering plays a key role in the biosynthesis of soybean isoflavones. Its main principle is to activate phenylpropanoid pathway genes by expressing maize C1 and R transcription factors in soybean seeds to increase the level of soybean isoflavones. Co-inhibition of flavanone 3-hydroxylase, which blocks the anthocyanin branch of the pathway, combined with C1/R expression can further increase the content of isoflavones (Yu et al., 2003). The R2R3-type MYB transcription factor GmMYB29 can regulate isoflavone biosynthesis by activating the promoters of key genes such as IFS2 and CHS8. As early as 2017, Chu et al. found that overexpression of GmMYB29 in soybean hairy roots can increase isoflavone content, while RNAi-mediated silencing leads to a decrease in isoflavone content. Feng et al. (2022) found that the C2H2-type zinc finger transcription factor GmZFP7 can affect the expression of gateway enzymes such as GmIFS2 and GmF3H1, and is a regulator of isoflavone accumulation.

8.2 CRISPR-based modifications to enhance isoflavone content

CRISPR/Cas9 technology provides a more precise method for modifying genes involved in isoflavone biosynthesis. Although specific studies on CRISPR-based modification in soybean to increase isoflavone content are limited, the technology has great potential. By targeting key regulatory genes (GmMYB29 and GmZFP7), CRISPR can be used to create knockouts or overexpressions to study the effects of key genes on isoflavone content. Chu et al. (2017) identified SNPs associated with isoflavone concentrations through genome-wide association studies (GWAS), providing valuable targets for CRISPR editing. In addition, transcriptome analysis can observe the dynamics of isoflavone biosynthesis genes during seed development, which can help researchers further understand potential CRISPR targets for increasing isoflavone content (Chen et al., 2023).

8.3 Synthetic biology for isoflavone production

Synthetic biology approaches have been widely used to reconstruct and optimize isoflavone biosynthesis in heterologous systems. In 2000, Jung et al. found that soybean isoflavone synthase could be successfully expressed in Arabidopsis to produce the isoflavone genistein, demonstrating the feasibility of transferring the pathway to other species. Researchers have also explored the concept of metabolons, protein complexes that promote efficient metabolic flux. In 2016, Dastmalchi et al. found that isoflavone synthase (IFS) and cinnamate 4-hydroxylase (C4H) can be anchored to the endoplasmic reticulum and interact with other enzymes in the pathway to form metabolons. This organization can improve the efficiency of isoflavone biosynthesis and provide a framework for synthetic biology applications. In addition, the identification of protein-protein interactions between isoflavone biosynthetic enzymes, such as GmIFS1 with GmCHS1 and GmCHIs, also further showed the potential to design these interactions to promote isoflavone production (Waki et al., 2016).

9 Conclusion and Future Directions

Isoflavones, plant secondary metabolites, have dual value in crop stress resistance and nutritional function. In recent years, important breakthroughs have been made in the biosynthesis of soybean isoflavones, and researchers have systematically analyzed the regulatory mechanism of its metabolic network. The dynamic assembly pattern of key enzyme complexes on the endoplasmic reticulum membrane has been revealed, and the synergistic mechanism of core catalytic elements such as isoflavone synthase (IFS) and chalcone synthase (CHS) has been elucidated. Genetic studies have identified key transcriptional regulatory factors such as GmMYB29 through genome-wide association analysis, confirming that they affect the activity of metabolic pathways through cascade regulatory networks. Gene editing strategies based on metabolic flux analysis have been successfully applied to seed isoflavone enrichment breeding, and targeted accumulation of target components has been achieved through competitive pathway inhibition and transcription factor co-expression technology.

Current research still faces bottlenecks in field transformation: First, isoflavone biosynthesis is regulated by the photoperiod and has developmental stage specificity, and field environmental fluctuations can easily lead to imbalances in the metabolic network; second, there are multi-level feedback regulations in the phenylpropanoid metabolic network, and genetic improvement of a single target may cause unexpected metabolic deviations. For example, although overexpression of IFS can increase the isoflavone content, it may change the distribution of lignin precursors, thereby affecting the mechanical strength of the plant. These complexities require researchers to establish a systems biology perspective and take into account both network homeostasis and target product output in metabolic engineering design.

Future research should focus on the following directions: (1) Integrate single-cell sequencing and spatial metabolomics technology to dynamically analyze the spatiotemporal specificity of isoflavone synthesis units during organ development; (2) Construct a mathematical model of metabolic network to simulate the laws of metabolic flux reprogramming under environmental disturbances and establish a precise and controllable genetic regulation strategy; (3) Develop a modular metabolic system based on synthetic biology to reduce bypass metabolic interference through compartmentalization engineering; (4) Combine intelligent agricultural equipment to establish a phenotype group-environment group association model and optimize cultivation management plans to stabilize the output of secondary metabolites. At the application level, the bioavailability and product stability of isoflavones can be improved through food processing technology innovations such as directed fermentation. Breakthroughs in these research directions will promote the actual transformation of isoflavones from basic research to agricultural quality improvement and functional food development.

Acknowledgments

Sincere thanks to every member of the project team for their hard work and sincere cooperation, as well as the valuable opinions and careful guidance of the reviewers, which have made this article perfect.

Conflict of Interest Disclosure

The author affirms that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

References

Barion G., Hewidy M., Panozzo A., Aloia A., and Vamerali, T., 2021, Effects of light orientation and mechanical damage to leaves on isoflavone accumulation in soybean seeds, Agronomy, 11(3): 589.

https://doi.org/10.3390/AGRONOMY11030589

Caldwell C., Britz S., and Mirecki R., 2005, Effect of temperature, elevated carbon dioxide, and drought during seed development on the isoflavone content of dwarf soybean [Glycine max (L.) Merrill] grown in controlled environments, Journal of Agricultural And Food Chemistry, 53(4): 1125-1129.

https://doi.org/10.1021/JF0355351

Chen H., Liu C., Li Y., Wang X., Pan X., Wang F., and Zhang Q., 2023, Developmental dynamic transcriptome and systematic analysis reveal the major genes underlying isoflavone accumulation in soybean, Frontiers in Plant Science, 14: 1014349.

https://doi.org/10.3389/fpls.2023.1014349

Chen X., Zhang K., Op G., D N., Kumar D., T V., A S., Gupta O., Nigam D., Dahuja A., Kumar S., Vinutha T., Sachdev A., and Praveen S., 2017, Regulation of isoflavone biosynthesis by mirnas in two contrasting soybean genotypes at different seed developmental stages, Frontiers in Plant Science, 8: 567.

https://doi.org/10.3389/fpls.2017.00567

Chennupati P., Seguin P., Chamoun R., and Jabaji S., 2012, Effects of high-temperature stress on soybean isoflavone concentration and expression of key genes involved in isoflavone synthesis, Journal of Agricultural and Food Chemistry, 60(51): 12421-12327.

https://doi.org/10.1021/jf3036319

Chu S., Wang J., Zhu Y., Liu S., Zhou X., Zhang H., Wang C., Yang W., Tian Z., Cheng H., and Yu D., 2017, An R2R3-type MYB transcription factor, GmMYB29, regulates isoflavone biosynthesis in soybean, PLoS Genetics, 13(5): e1006770.

https://doi.org/10.1371/journal.pgen.1006770

Dastmalchi M., Bernards M., and Dhaubhadel S., 2016, Twin anchors of the soybean isoflavonoid metabolon: evidence for tethering of the complex to the endoplasmic reticulum by IFS and C4H, The Plant Journal : For Cell and Molecular Biology, 85(6): 689-706.

https://doi.org/10.1111/tpj.13137

Feng Y., Zhang S., Li J., Pei R., Tian L., Qi J., Azam M., Agyenim-Boateng K., Shaibu A., Liu Y., Zhu Z., Li B., and Sun J., 2022, The dual-function C2H2-type zinc-finger transcription factor GmZFP7 contributes to isoflavone accumulation in soybean, The New Phytologist, 237(5): 1794-1809.

https://doi.org/10.1111/nph.18610

Gupta O., Dahuja A., Sachdev A., Jain P., Kumari S., T V., and Praveen S., 2019, Cytosine methylation of isoflavone synthase gene in the genic region positively regulates its expression and isoflavone biosynthesis in soybean seeds, DNA and Cell Biology, 38(6): 510-520.

https://doi.org/10.1089/dna.2018.4584

Gutierrez-Gonzalez J., Guttikonda S., Tran L., Aldrich D., Zhong R., Yu O., Nguyen H., and Sleper D., 2010, Differential expression of isoflavone biosynthetic genes in soybean during water deficits, Plant & Cell Physiology, 51(6): 936-948.

https://doi.org/10.1093/pcp/pcq065

Imaizumi R., Mameda R., Takeshita K., Kubo H., Sakai N., Nakata S., Takahashi S., Kataoka K., Yamamoto M., Nakayama T., Yamashita S., and Waki T., 2020, Crystal structure of chalcone synthase, a key enzyme for isoflavonoid biosynthesis in soybean, Proteins: Structure, 89: 126-131.

https://doi.org/10.1002/prot.25988

Jeong Y., An C., Park S., Pyun J., Lee J., Kim S., Kim H., Kim H., Jeong J., and Kim C., 2018, Methyl jasmonate increases isoflavone production in soybean cell cultures by activating structural genes involved in isoflavonoid biosynthesis, Journal of Agricultural and Food Chemistry, 66(16): 4099-4105.

https://doi.org/10.1021/acs.jafc.8b00350

Jung W., Yu O., Lau S., O'Keefe D., Odell J., Fader G., and Mcgonigle B., 2000, Identification and expression of isoflavone synthase, the key enzyme for biosynthesis of isoflavones in legumes, Nature Biotechnology, 18: 208-212.

https://doi.org/10.1038/72671

Kumar G., Saad K., Puthusseri B., Arya M., Shetty N., and Giridhar P., 2021, Exogenous serotonin and melatonin regulate dietary isoflavones profoundly through ethylene biosynthesis in soybean [Glycine max (L.) Merr.], Journal of Agricultural and Food Chemistry.

https://doi.org/10.1021/acs.jafc.0c07457

Kurosaki H., Koyano S., and Aoyama S., 2023, Effects of shading, soil moisture, fertilizations and sowing time on isoflavone content of soybean seed in Hokkaido, Plant Production Science, 26: 364-377.

https://doi.org/10.1080/1343943X.2023.2262755

Lozovaya V., Lygin A., Ulanov A., Nelson R., Daydé J., and Widholm J., 2005, Effect of temperature and soil moisture status during seed development on soybean seed isoflavone concentration and composition, Crop Science, 45: 1934-1940.

https://doi.org/10.2135/CROPSCI2004.0567

Mameda R., Waki T., Kawai Y., Takahashi S., and Nakayama T., 2018, Involvement of chalcone reductase in the soybean isoflavone metabolon: identification of GmCHR5, which interacts with 2‐hydroxyisoflavanone synthase, The Plant Journal, 96: 56-74.

https://doi.org/10.1111/tpj.14014

Matsuda H., Nakayasu M., Aoki Y., Yamazaki S., Nagano A., Yazaki K., and Sugiyama A., 2020, Diurnal metabolic regulation of isoflavones and soyasaponins in soybean roots, Plant Direct, 4: 056598.

https://doi.org/10.1101/2020.04.22.056598

Sarkar M., Watanabe S., Suzuki A., Hashimoto F., and Anai T., 2019, Identification of novel MYB transcription factors involved in the isoflavone biosynthetic pathway by using the combination screening system with agroinfiltration and hairy root transformation, Plant Biotechnology, 36(4): 241-251.

https://doi.org/10.5511/plantbiotechnology.19.1025a

Sohn S., Pandian S., Oh Y., Kang H., Cho W., and Cho Y., 2021, Metabolic engineering of isoflavones: an updated overview, Frontiers in Plant Science, 12: 670103.

https://doi.org/10.3389/fpls.2021.670103

Sugiyama A., Yamazaki Y., Hamamoto S., Takase H., and Yazaki K., 2017, Synthesis and secretion of isoflavones by field-grown soybean, Plant and Cell Physiology, 58: 1594-1600.

https://doi.org/10.1093/pcp/pcx084

Veremeichik G., Grigorchuk V., Butovets E., Lukyanchuk L., Brodovskaya E., Bulgakov D., and Bulgakov V., 2020, Isoflavonoid biosynthesis in cultivated and wild soybeans grown in the field under adverse climate conditions, Food Chemistry, 342: 128292.

https://doi.org/10.1016/J.FOODCHEM.2020.128292

Waki T., Yoo D., Fujino N., Mameda R., Denessiouk K., Yamashita S., Motohashi R., Akashi T., Aoki T., Ayabe S., Takahashi S., and Nakayama T., 2016, Identification of protein-protein interactions of isoflavonoid biosynthetic enzymes with 2-hydroxyisoflavanone synthase in soybean (Glycine max (L.) Merr.), Biochemical and Biophysical Research Communications, 469(3): 546-551.

https://doi.org/10.1016/j.bbrc.2015.12.038

Yang S.M., 2024, The role of microbial community structure in rice rhizosphere over the growing season, Genomics and Applied Biology, 15(5): 255-263.

Yu O., and Mcgonigle B., 2005, Metabolic engineering of isoflavone biosynthesis, Advances in Agronomy, 86: 147-190.

https://doi.org/10.1016/S0065-2113(05)86003-1

Yu O., Shi J., Hession A., Maxwell C., Mcgonigle B., and Odell J., 2003, Metabolic engineering to increase isoflavone biosynthesis in soybean seed, Phytochemistry, 63(7): 753-763.

https://doi.org/10.1016/S0031-9422(03)00345-5

Yuk H., Song Y., Curtis-Long M., Kim D., Woo S., Lee Y., Uddin Z., Kim C., and Park K., 2016, Ethylene induced a high accumulation of dietary isoflavones and expression of isoflavonoid biosynthetic genes in soybean (Glycine max) leaves, Journal of Agricultural and Food Chemistry, 64(39): 7315-7324.

https://doi.org/10.1021/ACS.JAFC.6B02543