MA and MB were soon acknowledged as potent allelochemicals derived from rice. Particularly, they exhibited substantial allelopathic activity against indicator plants, especially problematic weeds (e.g., Echinochloa crus-galli, Monochoria vaginalis, Solidago altissima, etc.) (Fukuta et al., 2007; Kato-Noguchi et al., 2013; Quan et al., 2019d), as well as against blast fungus (Magnaporthe oryzae) (Cartwright et al., 1980; Zhao et al., 2018a) (Table 1). Besides, MA and MB exerted inhibitory effects on the mycelia growth of some fungi causing plant diseases, including Botrytis cinerea (gray mold) (Fukuta et al., 2007), Fusarium solani (root rot, damping-off, and wilt) (Fukuta et al., 2007), Colletrotrichum gloeosporioides (anthracnose) (Fukuta et al., 2007), Fusarium oxysporum (Fusarium wilt) (Fukuta et al., 2007), Phytophthora infestans (blight) (Li et al., 2020), Colletotrichum acutatum (anthracnose) (Li et al., 2020), Ustilago maydis (maize smut) (Li et al., 2020), and Pyricularia oryzae (rice blast) (Li et al., 2020) (Table 1). Generally, MB displays stronger allelopathic activity compared to MA (Fukuta et al., 2007). The mixture of MA and MB (MAB) (1:1, w/w) demonstrated a similar inhibitory effect to MB against the germination rate and seedling growth of E. crus-galli and S. altissima (Quan et al., 2019d). Significantly, MA and MB had a negligible effect on rice growth, as reported by Kato-Noguchi et al. (2013).

Although MA and MB have shown high effectiveness in suppressing problematic weeds, neither of them has been successfully developed as a natural herbicide. This situation may be due to the limitations in the exploitation of natural momilactones, which can only be isolated in small amounts from plant sources (Kato et al., 1973; Chung et al., 2005; Nozaki et al., 2007; Liu et al., 2012; Ahmad et al., 2019; Quan et al., 2019d; Li et al., 2020). On the other hand, their allelopathic mechanisms have received relatively little scrutiny. According to Fang et al. (2015), MA and MB were proven to inhibit the growth of E. crus-galli and Arabidopsis through related gene expression and transcriptional pathways. In particular, MA and MB may inhibit the germination of Arabidopsis by suppressing the degradation process of cruciferin and cruciferina, which are storage proteins that play an important role in the germination process as the initial source of nitrogen (Kato-Noguchi et al., 2013). Besides, momilactones can inhibit Arabidopsis seedling growth, with Arabidopsis hypocotyls being more sensitive to MA and MB than roots (Kato-Noguchi et al., 2012). MB was notably stronger than MA in suppressing Arabidopsis seedling growth (Kato-Noguchi et al., 2012). The allelopathic mechanism of MB against Arabidopsis may be explained through the regulation of relevant proteins to the suppression of metabolic turnover and production of intermediators for cell structure (Kato-Noguchi and Kitajima, 2015). In another aspect, the absorption of momilactones by receiver plants has also been unclear. A previous report indicated the absorptive ability of momilactones by Arabidopsis, recording a similar absorption level of MA and MB (Kato-Noguchi et al., 2012). However, the absorption of momilactones by other indicator plants, especially problematic weeds (e.g., E. crus-galli, M. vaginalis, etc.) should be deeply understood to develop them as novel herbicides.

In preceding studies, comprehensive investigations on momilactone accumulation and release in the presence of competitive weeds were conducted through laboratory bioassays and practical field conditions (Kato-Noguchi et al., 2003; Kong et al., 2006). Moreover, the release levels of momilactones into the soil were estimated to be sufficient for inhibiting neighboring plants (Kato-Noguchi et al., 2003; Kong et al., 2006). However, the interactions between momilactones and environmental factors in the practical ecosystem have been least elucidated. In fact, the contents of allelochemicals in soils may be negatively affected through degradation, oxidation, binding to other substances, as well as soil conditions, comprising electrical conductivity, osmotic pressure, microorganisms (bacteria and fungi), and adsorption to soil particles (Tharayil et al., 2008; Yoneyama and Natsume, 2013; Anh et al., 2021a). Kong et al. (2006) announced that momilactone quantities released from rice straw into the soil gradually decreased during the decomposition process, especially no momilactones were detected after 3 months. In other reports, momilactone exudation into the root rhizosphere was regulated under biotic and abiotic stresses (weeds, UV, heavy metals, nutrient deficit, and cantharidin) (Kato-Noguchi, 2009). On the other hand, momilactone accumulation in donor plants and their secretion into the soil may be altered by the growth stage, biomass, and density of competitive plants. Therefore, the release of momilactones into the soil and their interaction with environmental factors should be extensively elaborated.

In another aspect, an effective approach is to develop allelopathic mixtures that can contribute to the development of herbicides with multiple action modes (Anh et al., 2021a). Currently, numerous allelochemicals have been individually acknowledged (e.g., momilactones, phenolics, terpenoids, and fatty acids, etc.) for their potent inhibition against weeds. However, the allelopathic interactions among these compounds have been rarely explored (Anh et al., 2021a). Naturally, allelochemicals tend to be released from host plants in mixture forms instead of single compound. Moreover, several studies have proven that mixtures reveal more potent suppressing impacts against tested plants compared to individual compounds (Quan et al., 2019d). For example, ME and 7-ketostigmasterol individually exhibited negligible effects on S. altissima (Quan et al., 2019d). However, their mixture dramatically diminished S. altissima growth (Quan et al., 2019d). This phenomenon may arise from the reciprocal and synergistic interactions of various compounds within a mixture, resulting in an effective inhibitory effect (Albuquerque et al., 2011; Anh et al., 2021a). Additionally, the synergistic interactions among compounds at the molecular level can regulate the allelopathic properties of the mixture (Albuquerque et al., 2011). Therefore, the combined application of momilactones and other promising allelopathic substances (e.g., phenolics, terpenoids, and fatty acids, etc.) is highly recommended to establish and explore their allelopathic effects and mechanisms (Anh et al., 2021a). Moreover, the impacts of practical conditions (e.g., recipient plant growth periods, density, uptakes, and environmental factors, etc.) on the activity of allelopathic mixtures should be thoroughly considered (Anh et al., 2021a). Furthermore, the dose-effectiveness of allelopathic mixtures is also an integral part of developing novel natural herbicides with high efficiency (Anh et al., 2021a).

Principally, a thorough understanding of the distribution of momilactones in plant sources will lead to effective approaches and strategies for their beneficial exploitation. Previously, momilactones were commonly found in cultivated rice (O. sativa L.) varieties. In the years 1973 and 1976, MA, MB, and MC were first discovered in rice husks (O. sativa L. var. Koshihikari) (Kato et al., 1973; Tsunakawa et al., 1976). In 2015, MD and ME were introduced for the first time, which were isolated from rice roots (O. sativa L. var. Chucheongbyeo) by Cho et al. (2015). However, ME was mistakenly classified, as explained in the introduction part (Figure 1). During the last few decades, momilactones have predominantly been detected in husks, leaves, and roots of various rice (O. sativa) cultivars. Recently, with improved techniques for detection and quantification, momilactones can be also found in different rice (O. sativa) organs (e.g., grains and brans) (Quan et al., 2019b). Chung et al. (2006) indicated that momilactones had higher amounts in Korean varieties, varieties with awns and late maturing time. Meanwhile, MA and MB quantities in japonica rice leaves were found to be higher than those in indica rice (Kariya et al., 2020). Lee et al. (1999) reported that momilactone contents in rice straws (O. sativa L. var. Haresugata) were increased to the highest levels at the heading stage and subsequently decreased.

Alongside cultivated rice, momilactones have also been detected in wild rice (Kariya et al., 2020). Specifically, species with AA genome (e.g., O. sativa, Oryza rufipogon, Oryza barthii, Oryza glaberrima, Oryza glumaepatula, and Oryza meridionalis) and BB genome (e.g., Oryza punctata) were reported to accumulate momilactones, whereas FF genome species (e.g., Oryza brachyantha) did not exhibit this accumulation. Notably, AA and BB genome species have a close phylogenetic distance, which are far from FF genome species (Kariya et al., 2020). Although the health-beneficial effects of MB are generally considered stronger than those of MA, most previous studies indicated lower levels of MB compared to MA in rice organs (Chung et al., 2006; Quan et al., 2019b; 2019c). For example, the content of MA was approximately twice that of MB in Koshihikari white grains (Quan et al., 2019b). In contrast, a recent investigation revealed that Koshihikari germinated brown rice showed a significantly higher level of MB than MA (Hasan et al., 2023), suggesting a potential source for MB exploitation that needs further investigation across different rice varieties. This finding may lead to proper and effective strategies in developing rice production and enhancing the consumption value of brown rice and rice-based products that benefit human health.

In moss (H. plumaeforme), MA and MB have been detected in aerial parts since 2007 (Nozaki et al., 2007). In 2020, MA, MB, and MF were purified from whole plant of H. plumaeforme (Li et al., 2020). Meanwhile, MA and MC have been found in aerial parts of another moss species (P. papillosa) (Liu et al., 2012).

Principally, the release of momilactones into the environment is a crucial research aspect pertaining to their role as allelochemicals contributing to the allelopathic activities of host plants. This aspect has been clearly demonstrated in numerous studies over several decades and comprehensively assembled and discussed in the report of Kato-Noguchi (2023), particularly focusing on rice plants. Based on these findings, the secretion of momilactones into the surrounding environment is emphasized in our work, as illustrated in Figure 3 for easier visualization. Furthermore, the exudation of momilactones from moss species is also debated in this study, which is in line with the release pattern of momilactones from rice plants under the influences of weed invasion and other unfavorable conditions. Despite the distinct genetic backgrounds of these two plant species, they exhibited similar response mechanisms to biotic and abiotic stresses regarding momilactone accumulation and release. This implies that momilactones might be important molecules playing a role in the general defense system of certain plant species. Further comprehensive studies should be conducted to validate this proposition. Especially, the release mechanism of momilactones from host plants under adverse conditions is a noteworthy topic that requires extensive elucidation.

In the research of Kato-Noguchi and Ino (2003), MB was secreted by rice seedlings at a sufficient phytotoxic level (1.8 nM/seedling), which inhibited cress (Lepidium sativum) in Petri dish. Additionally, the exudated levels of MB from seedlings were strongly correlated with the endogenous MB contents in rice seedlings. Therefore, the exudation of MB may depend on the endogenous production of MB in rice seedlings (Kato-Noguchi and Ino, 2005). In another report, 80-day-old rice plants exuded MB into their root rhizosphere at a concentration of 2.1 µg/plant/day, which was sufficient to suppress the growth of neighboring plants (Kato-Noguchi and Ino, 2004). Furthermore, Kato-Noguchi et al. (2003) indicated that MB was released during rice growth and development. The release levels rapidly increased to the maximal concentration in the flowering stage (2.1 µg/plant/day) and then sharply reduced (Figure 3). In a previous report, Kato-Noguchi et al. (2008) found that the endogenous content of MA was greater than that of MB, whereas the release of MB was higher than that of MA. Accordingly, Kato-Noguchi et al. (2008) hypothesized that MB was more selectively released into the root rhizosphere than MA. On average, an individual rice plant can release approximately 100 μg of MB into the surrounding environment throughout its growth duration (Kato-Noguchi et al., 2003). Theoretically, at a density of 100 rice plants/m², MB was estimated to be released into the soil with a content of 10 mg/m², which is sufficient to inhibit neighboring plants (Kato-Noguchi et al., 2003). According to Kong et al. (2006), MB was secreted into the soil by allelopathic rice varieties (PI312777 and Huagan-1) with a sufficient phytotoxic level at the early growth stage and achieved the highest concentration at the 5-leaf stage before dramatically reducing. Meanwhile, the non-allelopathic rice variety (Huajingxian) negligibly released MB into the root rhizosphere during its whole life cycle (Kong et al., 2006). Remarkably, the secreted content of MB by allelopathic rice was significantly enhanced (over 3 times higher) when they were grown with barnyardgrass (E. crus-galli) (Kong et al., 2006). However, this phenomenon was not observed in non-allelopathic rice grown in coculture with barnyardgrass (Kong et al., 2006). The results suggest that MB may significantly contribute to the rice defense system in the rhizosphere to compete with the invasion of neighboring plants (Kato-Noguchi et al., 2003). In another report, the allelopathic ability of rice plants may be increased through enhanced secretion of momilactones when rice was exposed to environmental stresses including UV, heavy metals, and cantharidin (Kato-Noguchi, 2009). In addition to root exudation, momilactones can be released into the soil through the decomposing process of rice straw (Figure 3), which inhibited surrounding plants (Kong et al., 2006).

Besides rice plants, moss (H. plumaeforme) can also secrete momilactones into the surrounding environment (Kato-Noguchi, 2011b). Interestingly, the endogenous amount of MA in H. plumaeforme was higher than that of MB; however, MB was released with a higher concentration than MA (Kato-Noguchi, 2011b). This suggests that MB is preferentially secreted by H. plumaeforme compared to MA, which aligns with the release propensity of momilactones in rice (Kato-Noguchi, 2011b). Furthermore, similar to rice, momilactones exudated from moss also increased under environmental stresses (UV, heavy metals, and cantharidin), leading to enhanced allelopathic activity (Kato-Noguchi, 2009). Accordingly, despite the phylogenetic distance between rice and moss, they may have a similar defense mechanism involving momilactone accumulation and release (Kato-Noguchi, 2011b).

To develop natural compounds as novel herbicides, understanding their degradation mechanisms is indispensable. Compound degradation involves processes of breakdown and transformation in the environment that influence their inherent activity, thereby being a significant reason for failures in practical applications. While the potential of momilactones for weed management has been extensively demonstrated, their degradation in the environment has not been thoroughly researched until now. Only one study in 2006 reported the levels of momilactones in the soil during different durations of rice straw decomposition (Figure 3). Specifically, momilactone amounts gradually reduced during this process (decreasing by over half from day 30 to day 60 for MB). Especially, MB was not detected in the soil after 90 days of decomposition (Kong et al., 2006). The study of Kong et al. (2006) implied that MB might undergo degradation in the soil within 90 days; however, its degradation mechanism remained unclear. Several factors can affect the degradation of natural compounds, consisting of temperature, pH, oxygen availability, enzyme catalysis, and microbial activity (Zhao et al., 2018b). Particularly, microorganisms (e.g., bacteria and fungi) play a crucial role in the degradation process through enzymatic activities, breaking down the compounds (Zhao et al., 2018b). Enzymatic reactions, such as oxidation and hydroxylation, can modify the structure of the compounds, resulting in their degradation (Zhao et al., 2018b). Therefore, these aspects are potential research directions to elucidate the degradation mechanisms of momilactones, which can significantly contribute to effective approaches and strategies for the practical implications of momilactones in weed management.

Sample preparation is a vital step in the isolation and purification processes of natural compounds (Anh et al., 2021a). Adequate sample preparation can enhance sample stability and extraction efficiency. Furthermore, careful preparation can help eliminate or minimize contaminants, supporting chemical analysis and subsequent isolation procedures (Palanisamy et al., 2020). The samples should be dried to reduce moisture content, as high moisture can lead to spoilage and microbial hydrolysis (Anh et al., 2023). In previous studies, Koshihikari rice husks were dried by sunlight for 1 week upon collection (Quan et al., 2019c). After that, the samples were meticulously sieved to eliminate impurities (Quan et al., 2019c). The samples were further dried in an oven at 50°C for 3 days (Quan et al., 2019c). Subsequently, the dried rice husks should be ground into powder to achieve homogeneity, facilitating easier and faster extraction using organic solvents. This physical processing aids in partially breaking down the cutin layer and cell wall, allowing for a swift release of compounds into the extraction solvent (Minh et al., 2018; Anh et al., 2023).

In the extraction steps for momilactones, the classical solid–liquid solvent extraction method (immersing dried rice or moss samples in organic solvents) has been commonly applied (Kato et al., 1973; Chung et al., 2005; Nozaki et al., 2007; Liu et al., 2012; Ahmad et al., 2019; Quan et al., 2019d; Li et al., 2020). Of which, methanol has been the most widely used solvent for this step (Chung et al., 2005; Ahmad et al., 2019; Quan et al., 2019d). Besides, aqueous methanol, acetone, and aqueous ethanol can also be employed (Nozaki et al., 2007; Liu et al., 2012; Li et al., 2020). While this conventional extraction method is simple, safe, and cost-effective (Garcia-Salas et al., 2010), it also has limitations such as time consumption, the need for large volumes of harmful solvents, and low efficiency (Garcia-Salas et al., 2010). Recently, only two studies aimed to improve the extraction process for momilactones (Minh et al., 2018; Ahmad et al., 2019). In particular, Minh et al. (2018) established a simple and optimized process for extracting MA and MB from rice husks by combining EtOAc, MeOH, distilled water, temperature, and pressure. The results demonstrated that rice husks treated with heat (100°C) and extracted using a combination of EtOAc and MeOH yielded the highest quantities of MA and MB (Minh et al., 2018). Besides, MB was obtained with larger amounts compared to MA (Minh et al., 2018). The application of pressure also enhanced the yields of MA and MB, resulting in respective 5- and 15-fold increases in purified amounts (Minh et al., 2018). In the study of Ahmad et al. (2019), different techniques for momilactone extraction, including percolation, agitation with heat, sonication, and Soxhlet, were examined, which screened various solvent systems, comprising EtOAc, acetone, acetonitrile, MeOH, and MeOH:water (8:2, v/v). Accordingly, the maximum extraction efficiency was achieved when applying a mixture of MeOH and MeOH:water in Soxhlet extraction, surpassing other methods (Ahmad et al., 2019). Besides, the applied processes of defatting under low temperature (Ahmad et al., 2019; Quan et al., 2019c), mixing with activated charcoal, filtering with celite beds (Ahmad et al., 2019), and Sep-Pak C18 cartridges (Quan et al., 2019d) during the treatment of crude extracts can aid in removing unwanted components, leading to higher yields and purer momilactones. Furthermore, advanced methods including microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), supercritical CO₂ extraction (SC-CO₂), pressurized fluid extraction (PFE), and enzyme-assisted extraction (EAE) have shown high effectiveness in extracting natural compounds from plant sources (Aires, 2017). These techniques should be examined in further investigations for momilactone extraction to establish effective methods that help overcome the disadvantages of classical protocols and enhance efficiency.

In the next step of momilactone isolation, the obtained crude extracts can be dissolved in water and fractionated using liquid–liquid phase separation with different solvents (e.g., hexane and EtOAc) (Table 3; Figure 4). Subsequently, fractions containing momilactones can be collected for further isolation and purification processes. The most common method for momilactone isolation is column chromatography (Kato et al., 1973; Chung et al., 2005; Nozaki et al., 2007; Liu et al., 2012; Ahmad et al., 2019; Quan et al., 2019d; Li et al., 2020), which can separate compounds based on their adsorption (Palanisamy et al., 2020).

To support the isolation process, widely applied methods for the analysis of momilactones in obtained extracts and fractions include thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), high resolution-mass spectrometry (HR-MS), electron ionization-mass spectroscopy (EI-MS), fast atom bombardment-mass spectrometry (FAB-MS), infrared spectroscopy (IS), electrospray ionization-mass spectrometry (ESI-MS), Fourier transform-infrared spectroscopy (FT-IR), high resolution-electrospray ionization-mass spectrometry (HR-ESI-MS), high-performance liquid chromatography-photo diode array-mass spectrometry (HPLC-PDA-MS), nuclear magnetic resonance (NMR), and X-ray crystallization (Table 3).

Among the aforementioned methods, TLC, with the advantages of simplicity, time- and cost-effectiveness, has been widely applied for momilactone identification in obtained fractions. As described by Quan et al. (2019b), TLC analysis for momilactones can be conducted on Merck TLC silica gel 60 plates with a layer thickness of 175–225 μm. MA and MB standards were used as references (Quan et al., 2019b). The fractions containing MA and MB were spotted onto the TLC plate and run alongside a mobile phase of CHCl₃:MeOH (9.5:0.5, v/v) for 2.4 min (Quan et al., 2019b). The TLC plate was then stained with 1% vanillin-sulfuric acid in pure EtOH and dried in an oven at 100°C for 2 min. The separation of MA and MB was visually observed (Quan et al., 2019b) (Figure 5A). The retention factor (R_f) values were also calculated for the confirmation of MA and MB, based on the ratio of the traveled distance of the MA or MB spot to the traveled distance of the solvent on the TLC plate (Quan et al., 2019b).

HPLC analysis is another effective approach for identifying momilactones. In which, isolated momilactones can be dissolved in MeOH and subjected to an HPLC system with a mobile phase consisting of 0.1% trifluoroacetic acid in 70% acetonitrile (Quan et al., 2019b). The analysis is operated with a detector set at 210 nm and a flow rate of 0.4 mL/min over 30 min (Quan et al., 2019b). As a result, isolated MA and MB are detected with retention times (RT) of 17.03 and 14.06 min, respectively, in comparison to standards (Figure 5B) (Quan et al., 2019b).

For molecular mass characterization, isolated MA and MB can be confirmed by GC-MS method, as presented in the study of Quan et al. (2019c). In which, MA and MB were identified with molecular ions at 314 and 330 m/z, respectively (Figure 5C) (Quan et al., 2019c). Additionally, the molecular masses of MA and MB can be confirmed using ESI-MS in a positive mode, as described by Hasan et al. (2023). According to the results, MA and MB were characterized with m/z = 315.19470 and 331.19006 [M + H]⁺, respectively (Figure 5D) (Hasan et al., 2023). The data obtained from GC-MS and ESI-MS were compared to literature and standards to confirm the isolated MA and MB (Quan et al., 2019c; Hasan et al., 2023).

The structures of isolated momilactones can be determined applying NMR method, which has been included in numerous studies. For instance, the ¹H- and ¹³C NMR spectra of MA and MB (in deuterated chloroform, CDCl₃) were acquired on an NMR spectrometer at 600 and 151 MHz, respectively (Hasan et al., 2023). Examples of the ¹H- and ¹³C NMR spectra of MA and MB are presented in Figure 5E. The NMR spectra of isolated MA and MB were confirmed by comparing to literature (Hasan et al., 2023).

Hitherto, several techniques have been developed for quantifying momilactones in plant samples. Among these, HPLC has been commonly applied, being a central focus of development. However, due to the minor amounts of momilactones present in the plant sources, their detection and quantification have faced numerous limitations and challenges. To determine momilactone profiles in plant extracts using HPLC, the extraction process demands multiple complex steps to purify the extract and eliminate unwanted and interfering components (Kato-Noguchi and Kobayashi, 2009). Most studies have quantified momilactones in husks, leaves, and roots (Lee et al., 1999; Xuan et al., 2016; Minh et al., 2018; Quan et al., 2019d). Specifically, these methods employed columns with lengths ranging from 150 to 200 mm and an isocratic mobile phase (e.g., 0.1% trifluoroacetic acid in 70% aqueous acetonitrile) with a flow rate ranging from 0.4 to 0.8 mL/min. The procedures required a total run time of around 1 h per sample (Table 4) (Kato-Noguchi and Kobayashi, 2009; Minh et al., 2018), leading to time and solvent consumption. Recently, an improved ultra-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS) method has been generated applying a 50 mm column alongside a gradient mobile phase consisting of 0.1% trifluoroacetic acid in water and 0.1% trifluoroacetic acid in acetonitrile with a flow rate of 0.3 mL/min, which reduced the total run time to 15.1 min for each sample (Quan et al., 2019a). This method did not require complicated extraction steps (Quan et al., 2019c; Anh et al., 2022b; Hasan et al., 2023; Jesmin et al., 2023). Notably, the protocol has demonstrated effectiveness and high detection sensitivity in identifying and quantifying momilactones in extract samples with low concentrations and complex mixtures containing various substances (Quan et al., 2019c; Anh et al., 2022b; Hasan et al., 2023; Jesmin et al., 2023). Furthermore, by examining a blank sample (e.g., pure MeOH) after measurements of each sample, no contamination and interfering chemicals were recorded, resulting in high analysis accuracy (Quan et al., 2019c; Anh et al., 2022b; Hasan et al., 2023; Jesmin et al., 2023). One of the breakthroughs of this method is its first-time detection and quantification of MA and MB in rice grains and brans, despite their presence in minor amounts (Quan et al., 2019c). By applying this protocol, MA were found with the highest quantity in rice husks (16.44 μg/g DW), followed by rice roots (8.06 μg/g DW), brans (6.65 μg/g DW), leaves (4.28 μg/g DW), and grains (2.07 μg/g DW) (Quan et al., 2019b). While MB were determined with the highest content in rice leaves (12.73 μg/g DW), followed by husks (9.24 μg/g DW), brans (6.24 μg/g DW), roots (5.69 μg/g DW), and grains (1.06 μg/g DW) (Quan et al., 2019b). An example of UPLC-ESI-MS analysis for confirmation and quantification of MA and MB in germinated brown rice samples is presented in Figure 6 (Hasan et al., 2023), in which peak detection and areas are shown.

In identification and quantification methods for natural compounds using HPLC techniques, the values of limit of detection (LOD) and limit of quantification (LOQ) are also important parameters, indicating the specific accuracy and reliability of the methods. Quan et al. (2019d) explained that there are several principal factors that contribute to enhancing the efficiency of HPLC analysis. For instance, the utilization of a Sep-Pak C18 cartridge in preparing extract samples can aid in the removal of unwanted or interfering substances, thereby yielding purer MA and MB. In addition, adjusting the conditions of the stationary and mobile phases and the flow rate can also affect the sensitivity of HPLC system. Particularly, employing a 150 mm column with an isocratic mobile phase (0.1% TFA in 70% aqueous acetonitrile) at a flow rate of 0.5 mL/min over 25 min demonstrated highly effective LOD (0.05 ng/mL) and LOQ (0.14 ng/mL) values for the detection and quantification of MA (Quan et al., 2019d). On the other hand, the UPLC-ESI-MS method, utilizing a 50 mm column with a gradient mobile phase (0.1% trifluoroacetic acid in water and 0.1% trifluoroacetic acid in acetonitrile) at a flow rate of 0.3 mL/min over 15.1 min, showed impressive sensitivity in detecting and quantifying MB with LOD and LOQ of 0.27 and 0.83 ng/mL, respectively (Quan et al., 2019a).

In preceding studies, the momilactone biosynthetic pathway (MBP) was extensively discussed (Nguyen and Dang, 2021; Serra et al., 2021; Kato-Noguchi, 2023). Specifically, Kato-Noguchi (2023) and Serra et al. (2021) predominantly focused on the MBP in rice and its association with allelopathic activity. On the other hand, Nguyen and Dang (2021) highlighted and deliberated the achievements obtained in unraveling the MBP over the past five decades since the first discovery of momilactones, outlining limitations and proposing promising strategies to further clarify the remaining gaps. For our study, understanding the MBP is pivotal which may enable effective approaches to enhance momilactone production in plant sources, thereby facilitating the exploitation of these compounds. Accordingly, we delve into the MBP along with the relevant enzymes and their specific functions in the formation of momilactones from precursors (Figure 7). Notably, we discuss the MBP across various plant species such as rice and moss. Factors and environmental conditions influencing momilactone biosynthesis are also presented in our research (Figure 7). Based on that, momilactones produced by the host plant may be regulated through the adjustment of external conditions. Furthermore, research on improving plant tolerance to stresses through the MBP is considered and proposed.

In rice (O. sativa), momilactone biosynthetic genes (MBGs) have been located in the 168-kb region of the fourth chromosome (Figure 6), which includes OsCPS4, OsKSL4, CYP99A2, CYP99A3, and OsMAS genes (Wang et al., 2011; NCBI, 2023). These genes encode the enzymes consisting of syn-copalyl diphosphate synthase-like, syn-pimara-7,15-diene synthase-like, cytochrome P450 99A2-like, 9-beta-pimara-7,15-diene oxidase-like, and momilactone A synthase-like, respectively (Wang et al., 2011; NCBI, 2023). Particularly, the roles of CYP99A2 and CY99A3 in the MBP were confirmed in previous studies (Shimura et al., 2007; Wang et al., 2011). In which, CYP99A2 was found to have less involvement in this pathway compared to CYP99A3. These two enzymes act on the oxidation of the carbon-19 (C19) methyl group of (9β-H)-pimara-7,15-diene to form (9β-H)-pimara-7,15-dien-19-oic acid (Shimura et al., 2007), a predicted precursor of MC and ME (Zhao et al., 2018a). Subsequently, the lactone ring in the 3β-hydroxy-(9β-H)-pimara-7,15-dien-19,6β-olide structure is generated from (9β-H)-pimara-7,15-dien-19-oic acid via hydroxylation at the C3β- and C6β-positions with the catalytic activities of P450 genes CYP701A8 and CYP76M8 (Wang et al., 2012). In the formation of MA, two short-chain alcohol dehydrogenases/reductases (SDRs) catalyze the oxidation of 3β-hydroxy-(9β-H)-pimara-7,15-dien-19,6β-olide to produce the keto group of MA at the C3-position (Shimura et al., 2007; Kitaoka et al., 2016). Based on that, these two SDRs have been defined as momilactone synthase (MS) (Shimura et al., 2007; Kitaoka et al., 2016). For MB biosynthesis, hydroxylation at the C20-position and hemiketal ring closure are required (Zhang and Peters, 2020); however, the enzymes and related genes involved in this process have not been characterized yet.

A similar situation has been found in the momilactone biosynthetic gene cluster (MBGC) of other species within the Poaceae family, which is correlated with their close phylogenetic relationships (Miyamoto et al., 2016). For instance, O. punctata has been found to accumulate momilactones and show an analogous MBGC to that of O. sativa (Miyamoto et al., 2016). On the other hand, O. brachyantha and Leersia perrieri, which have more phylogenetic distance, neither produce momilactones nor contain the relevant biosynthetic genes (Miyamoto et al., 2016). Miyamoto et al. (2016) concluded that the MBGC evolved within the Oryza lineage. Remarkably, E. crus-galli, with much more phylogenetic distance from Oryza lineage, also possesses an orthologous MBGC (Guo et al., 2017). Phylogenetic analysis indicated that the MBGC of E. crus-galli is more consistent with that of Oryza than with other closely related plant species (Guo et al., 2017). However, the MBGC of E. crus-galli contains single copies of the CYP99A and MS families with some modifications in gene order (Guo et al., 2017). Additionally, E. crus-galli has not been reported to produce endogenous momilactones. This suggests that no momilactone relevant biosynthetic gene is self-sufficient.

Another species that both contains an MBGC and produces momilactones is C. plumiforme (moss) (Mao et al., 2020). Based on the genome sequence, the previously reported diterpene cyclase CpDTC was located (Okada et al., 2016), revealing that the neighboring region contains two specific CYPs (CYP75B and CYP707C subfamilies) and an SDR. Remarkably, CYP75B plays a role in C19 oxidase activity, similar to the Poaceae CYP99A (Okada et al., 2016). Besides, the C. plumiforme SDR was reported to have the same catalytic activity as the Poaceae MS (Okada et al., 2016). On the other hand, CYP707C was found to act as a C3β-hydroxylase (Okada et al., 2016). Accordingly, the two separately evolved MBGCs in rice and moss exhibited exactly the same relevant enzymatic activities. This leads to questions about these analogies despite a distinct evolutionary distance that requires deeper clarification.

Since the complete steps of the catalytic processes in momilactone biosynthesis have been unclear, especially the conversion from MA to MB. de La Peña and Sattely (2021) developed an effective approach by screening Nicotiana benthamiana genes, which clarified the entire pathway for momilactone biosynthesis, resulting in a yield over 10 times higher than that observed in rice.

Momilactones were first identified as rice allelochemicals. Accordingly, Kato-Noguchi (2011a) indicated that the MB content in rice seedlings grown with barnyardgrass was 6.9 times higher than that of rice plants grown independently. Besides, momilactone proliferation was recorded in rice response to pathogen attack (Umemura et al., 2003), UV (Kato-Noguchi, 2009; Anh et al., 2022b), heavy metals (Kato-Noguchi, 2009), salinity (Xuan et al., 2016; Huong et al., 2020; Xuan et al., 2022; Jesmin et al., 2023), drought (Xuan et al., 2016), chilling (Anh et al., 2022b), nutrient deficit (Kato-Noguchi, 2009), cantharidin (Kato-Noguchi, 2009), and jasmonic acid (JA) (Kato-Noguchi, 2009). These changes in rice momilactone contents can be explained through the expression of relevant genes in the biosynthetic pathway (Bajsa-Hirschel et al., 2020). For example, the expression of momilactone biosynthetic genes (MBGs), especially OsMAS and OsMAS2, was significantly elevated in the presence of banryardgrass (Bajsa-Hirschel et al., 2020). In another study, the production of momilactones in rice was enhanced under UV irradiation, consistent with the upregulated expression of MBGs (Anh et al., 2022b). Conversely, the accumulation of momilactones and expression of MBGs decreased under chilling effects (Anh et al., 2022b). Nevertheless, the impact of other adverse conditions (e.g., salinity, drought, heavy metals, nutrient deficit, etc.) on MBGs has not yet been elucidated.

In moss, momilactone accumulation also increased when exposed to barnyardgrass, heavy metals, and cantharidin (Kato-Noguchi and Kobayashi, 2009; Kato-Noguchi, 2011b). Momilactone contents in moss under the influence of biotic and abiotic stresses may also be regulated through the expression of related biosynthetic genes. For example, HpDTC1, which encodes a diterpene cyclase involved in the conversion of geranylgeranyl diphosphate (GGDP) to syn-pimara-7,15-diene in moss, showed an upregulation in expression when moss was subjected to stresses (Okada et al., 2016).

Momilactones are also known as labdane-related diterpenoids, sharing the same initiator (GGDP) with gibberellic acid (GA), a phytohormone, in the biosynthetic pathway. In particular, these compounds are formed through consecutive catalysis by two distinct diterpene synthases and cyclases (Miyamoto et al., 2016). Notably, GA has been identified as a major contributor to plant resistance against abiotic stresses, including cold, salt, osmotic, and submergence conditions (Colebrook et al., 2014). Meanwhile, momilactones have been proven to be responsive compounds in rice and moss plants against environmental stresses, including weed invasion (Kato-Noguchi, 2011b; 2011a; Bajsa-Hirschel et al., 2020), pathogen attack (Umemura et al., 2003), UV (Kato-Noguchi, 2009; Anh et al., 2022b), heavy metals (Kato-Noguchi, 2009), salinity (Xuan et al., 2016; Huong et al., 2020; Xuan et al., 2022; Jesmin et al., 2023), drought (Xuan et al., 2016), chilling (Anh et al., 2022b), nutrient deficit (Kato-Noguchi, 2009), and cantharidin (Kato-Noguchi, 2009).

The production of momilactone may be motivated by jasmonic acid (JA)-dependent and independent signaling pathways (Kato-Noguchi, 2023). For instance, after CuCl₂ application, the involvement of JA biosynthesis inhibitors, comprising quinacrine, nordihydroguaiaretic acid, and salicylhydroxamic acid, prevented MA accumulation in rice (Rakwal et al., 1996). However, the subsequent addition of exogenous JA resulted in the proliferation of endogenous MA in rice (Rakwal et al., 1996), indicating that JA stimulated MA biosynthesis. Apart from JA, the exogenous application of salicylic acid (SA) also promoted momilactone accumulation in rice, though the exact mechanism remains unclear (Daw et al., 2008). Notably, JA and SA are signaling phytohormones that trigger plant defense responses, consisting of various defensive reactions to safeguard the plant (Zhao et al., 2017; Howe et al., 2018; Nguyen et al., 2022). In another report, Xuan et al. (2016) indicated that MA and MB were involved in rice response to abiotic stresses, including drought and salinity, rather than weed resistance. The accumulation of MA and MB in rice was also increased by the exogenous application of fulvic acid and magnesium sulfate (MgSO₄) during salinity conditions, in line with rice’s elevated tolerance to salt stress (Xuan et al., 2022; Jesmin et al., 2023). These pieces of evidence suggest that momilactones might play essential roles in the plant’s defense system against adverse environments. However, their actual contribution remains elusive. Remarkably, Anh et al. (2022b) suggested that momilactones might not directly contribute to the antioxidant response of rice against UV and chilling stresses. Instead, a concomitant biosynthesis between momilactones and some acknowledged stress-resistant phenolics was documented in UV- and chilling-exposed rice plants (Anh et al., 2022b). In another research, the biosynthesis of momilactones was consistent with that of GA in rice cocultured with barnyardgrass (Bajsa-Hirschel et al., 2020). Consequently, we hypothesize that momilactones may act as signaling molecules in rice physiological response to unfavorable conditions.

Another consideration is that although moss MBGC differs from rice MBGC in terms of gene sequences and orders, they may share a similar response mechanism against environmental stresses with respect to momilactone biosynthesis and relevant gene expressions, as described in the previous section. Therefore, a proposed question arises: may momilactones play a role as novel phytohormones in rice and moss? This requires comprehensive validation in future studies. On the other hand, the potential signaling mechanisms of momilactones and their relationship with resistance-associated compounds (e.g., phytohormones: GA, SA, abscisic acid, ethylene, and JA) in rice and moss under stresses, as well as functional enzymes (e.g., antioxidant enzymes and DNA damage repair enzymes), may be promising fields of study that need further scrutinization.