WT Nipponbare rice plants were grown under controlled conditions (a 12 h photoperiod, 200-µmol photons m⁻² s⁻¹ and day/night temperature of 27/25°C). Rice seeds were surface-sterilized in a 50% sodium hypochlorite solution with 0.01% Tween-20 for 15 min. Seeds were then rinsed with sterile water and germinated in the dark overnight. The pre-germinated seeds were transferred to Petri dishes containing half-strength liquid Murashige and Skoog (MS) medium and incubated in a growth chamber for 7 days. Thereafter, the seedlings were transferred into black falcon tubes filled with low-Pi of half-strength modified Hoagland nutrient solution with adjusted pH to 5.8. The nutrient solution consisted of 5.6 mM NH₄NO₃, 0.8 mM MgSO₄·7H₂O, 0.8 mM K₂SO₄, 0.18 mM FeSO₄·7H₂O, 0.18 mM Na₂EDTA·2H₂O, 1.6 mM CaCl₂·2H₂O, 0.8 mM KNO₃, 0.023 mM H₃BO₃, 0.0045 mM MnCl₂·4H₂O, 0.0003 mM CuSO₄·5H₂O, 0.0015 mM ZnCl₂, 0.0001 mM Na₂MoO₄·2H₂O and 0.004 mM K₂HPO₄·2H₂O.

Detailed synthetic method is available in Supplementary Document 1 .

For phenotyping, 1 week-old rice seedlings were transferred into 50 mL tubes, filled with half-strength modified Hoagland nutrient solution with 5.8 pH. Seedlings were treated with 1.0 µM MiZax (solved in 0.1% acetone) or the corresponding volume of the solvent (control) for two weeks. The solution was refreshed at two days intervals.

The SL analysis in rice root exudates was performed according to the protocol published in (Wang et al., 2022c). For this purpose, rice plants were grown hydroponically in 50 mL tubes for two weeks under low phosphate conditions and treated with 5.0 μM MiZax (solved in 0.1% acetone) or the corresponding volume of the solvent (control) for 6 h. SLs were then collected from root exudates. Briefly, 50 mL of root exudates spiked with 0.672 ng of 20 ng rac-GR24 was brought on a C₁₈-Fast Reversed-Phase SPE column (500 mg 3 mL^-1) preconditioned with 3 mL of methanol and 3 mL of water. After washing with 3 mL of water, SLs were eluted with 5 mL of acetone. The SLs fraction was concentrated to SL aqueous solution (∼1 mL), followed by 1 mL of ethyl acetate extraction. 750 μL of SL enriched organic phase was dried under vacuum. The final extract was re-dissolved in 100 μL of acetonitrile: water (25:75, v:v) and filtered through a 0.22 μm filter for LC-MS/MS analysis.

SLs were quantified by LC-MS/MS using a UHPLC- Triple-Stage Quadrupole Mass Spectrometer (Thermo Scientific™ Altis™). Chromatographic separation was the same as above SL identification. The MS parameters were: positive ion mode, ion source of H-ESI, ion spray voltage of 5000 V, sheath gas of 40 arbitrary units, aux gas of 15 arbitrary units, sweep gas of 2 arbitrary units, ion transfer tube gas temperature of 350°C, vaporizer temperature of 350 °C, collision energy of 17 eV, CID gas of 2 mTorr. The characteristic Multiple Reaction Monitoring (MRM) transitions (precursor ion → product ion) were 331.15→216.0, 331.15→234.1, 331.15→97.02 for 4-deoxyorobanchol; 347.14→329.14, 347.14→233.12, 347.14→ 205.12, 347.14→97.02 for orobanchol; 361.16→ 247.12, 361.16→177.05, 361.16→208.07, 361.16→97.02 for 4-oxo-MeCLA; 299.09→185.06, 299.09→157.06, 299.09→97.02 for GR24. 317.17→ 220.14, 317.17→205.12, 317.17→164.08, 317.17→97.02 for CL+14 (putative oxo-CL).

Assays were performed as the published procedure (Jamil et al., 2023). Rice plants were grown hydroponically in 50 mL tubes for two weeks under low-phosphate conditions and treated with 5.0 µM MiZax for 6 h. SLs were extracted from root exudates using C18 columns and applied to pre-conditioned Striga seeds. For pre-conditioning, Striga seeds were surface-sterilized with 50% bleach for 5 min, rinsed six times with sterile Milli-Q water, and air-dried. Approximately 50–100 seeds were placed on 9 mm glass fiber filter paper discs. Twelve discs were transferred to Petri dishes containing moistened Whatman paper, sealed, and incubated at 30°C in darkness for 10 days. The pre-conditioned Striga seeds were treated with 55 µL of each sample and incubated at 30°C for 24 h. Germinated and total seeds were counted using SeedQuant (Braguy et al., 2021) to calculate germination percentages.

Data are represented as mean and their variations as standard deviation. The statistical significance was determined by one-way analysis of variance (one-way ANOVA) with Tukey’s multiple comparison test, using a probability level of p<0.05; or two-tail student t-test with denote significant differences (*p < 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001). All statistical elaborations were performed using GraphPad Prism, version 8.3.0.

To investigate the impact of structural modifications to the methoxybenzene ring on biological activity, we designed and synthesized compounds MZ9 through MZ12. In these analogs, the para-methoxy group of MZ3 was substituted with a fluoro, ethyl, meta-methoxy, or ortho-methoxy group, respectively ( Figure 1B ). To assess the activity of the new mimics, we used MZ3 as reference, which showed promising effects at low concentrations under both hydroponic (1.0 µM) and open-field conditions (2.5 µM) (Wang et al., 2020; Wang et al., 2022a; Wang et al., 2023b; Jamil et al., 2023), we tested the new mimics at these concentrations. We, evaluated their growth-promoting effects at a concentration of 1.0 µM on hydroponically grown rice seedlings, using MZ3 as a positive control. All four compounds exhibited significant growth-enhancing activity, leading to a 30% to 50% increase in root length and a 10% to 20% increase in crown root number ( Figures 2B, C ). Notably, only MZ10 significantly improved shoot biomass ( Figure 3C ), showing the most pronounced overall effect with an approximately 20% increase in crown root number and a 40% enhancement in shoot biomass.

Moreover, it is worth noting that the growth-promoting effects of these compounds were more pronounced in roots than in shoots, consistent with previous findings on earlier generations of MiZax (Wang et al., 2020; Jamil et al., 2023). The positive control, MZ3, displayed comparable activity to MZ9–MZ12 in promoting root length, crown root number, shoot length, and tiller development ( Figures 2 , 3 ).

We recently reported the growth-promoting effects of zaxinone and its synthetic mimics, MiZax, with a particular emphasis on root growth and development in rice (Wang et al., 2020; Jamil et al., 2023). The promising outcomes from these studies highlight the potential utility of these biostimulants for field applications aimed at improving several crop performance and productivity (Wang et al., 2020, 2022a; Wang et al., 2023b; Jalal et al., 2025). Previous studies showed that substituting the ketone group of MZ3 with imine, alcohol, or ether moieties enhanced root biomass without causing adverse effects, suggesting that while the ketone contributes to bioactivity, its position allows for a degree of structural flexibility (Jamil et al., 2023). In the present study, we developed a new series of MiZax by introducing structural modifications to the methoxybenzene ring of MiZax3, aiming to investigate their impact on biological activity.

The substitution of the para-methoxy group in MZ3 with fluoro, ethyl, meta-methoxy, or ortho-methoxy groups resulted in similar positive effects on root growth and an increase in the number of crown roots in rice seedlings, comparable to MZ3. This suggests that the “-O-” group and the position of the methoxy group do not significantly restrict bioactivity. Notably, the ethyl substitution (MZ10) exhibited slightly enhanced activity compared to MZ3, particularly in regulating crown root development and shoot biomass. This suggests that elongation on the alkyl chain could enhance the growth-promoting activity.

Next, we assessed the effects of MZ9–MZ12 on SL content in rice root exudates, which is usually measured after a 6-hour incubation period (Wang et al., 2019; Ito et al., 2022; Jamil et al., 2023). Treatment with these compounds at a 5.0 µM concentration led to a significant reduction in SL levels, as confirmed by LC–MS analysis Specifically, the canonical SL orobanchol levels decreased by 20% to 50%, while methyl 4-oxo-carlactonoate (4-oxo-MeCLA), a non-canonical SL, was reduced by 34% to 78%. In contrast, 4-deoxyorobanchol, the second major canonical SL in rice, was significantly suppressed only by MZ9. Notably, none of the compounds markedly affected the levels of tentative oxo-carlactone (oxo-CL) (Wang et al., 2022b) ( Figure 4A ). The effects were consistently validated through a Striga bioassay, which demonstrated a reduction of approximately 30% in Striga seed germination compared to the blank treatment ( Figure 4B ). Similarly, the positive control, MZ3, resulted in a reduction of approximately 45% in orobanchol levels, 80% in 4-deoxyorobanchol, 85% in 4-oxo-MeCLA, and 40% in Striga seed germination ( Figures 4A, B ).

The apocarotenoid hormone SLs not only regulates developmental processes that shape shoot and root architecture but also facilitate the colonization of host plants by symbiotic fungi (Al-Babili and Bouwmeester, 2015; Lanfranco et al., 2018). However, the quantity and composition of SLs released by host roots into the rhizosphere are directly related to the infection rate of root parasitic weeds (Wang et al., 2024a). Therefore, reducing the secretion of SLs could potentially lower the infection rates of root parasitic plants in the rhizosphere (Kuijer et al., 2024). In our study, treatment with MZ9–MZ12 generally led to a reduction in the levels of various SLs, particularly orobanchol and 4-oxo-MeCLA, while having minimal impact on the amount of oxo-CL. However, most of the reduced SLs in this study are weak germination stimulants for Striga. Indeed, several studies have demonstrated that 4-deoxyorobanchol is a stronger germination stimulant for Striga seeds than orobanchol and 4-oxo-MeCLA in rice (Ito et al., 2022; Chen et al., 2023; Li et al., 2024). Notably, the suppressive effects of MiZax on SL levels varied depending on the specific SL type affected, suggesting that certain MiZax may be particularly valuable not only for mitigating Striga infection but also for modulating beneficial arbuscular mycorrhizal symbiosis (Lanfranco et al., 2018; Wang et al., 2024a). In this study, we observed that the fluoro substitution in the methoxybenzene ring (MZ9) significantly decreased the level of 4-deoxyorobanchol without impacting the downstream metabolite orobanchol. Conversely, substitutions with ethyl, meta-methoxy, or ortho-methoxy groups (MZ10–12) did not affect 4-deoxyorobanchol levels but led to a suppression of orobanchol accumulation. These findings suggest that the functional groups present on the methoxybenzene ring may selectively affect the expression or the enzymatic activity of MAX1–900 or MAX1-1400 ( Figure 1A ). Further investigations are warranted to validate this hypothesis.

Taken together, our results further demonstrate that structural modifications to the methoxybenzene ring of the previously developed MiZax3 did not diminish bioactivities, particularly in promoting rice growth. This study thus provides valuable insights to inform future chemical design and highlights potential targets for research on SL-related pathways as well as zaxinone biology.