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The discovery and beneficial use of natural products can help improve human life. Plant growth inhibitory chemicals are widely used as herbicides to control weeds. Due to the need to use different types of herbicides, there is a need to identify compounds with new mechanisms of action. In this study, we discovered a novel N -alkoxypyrrole compound, coumamonamide, from Streptomyces werraensis MK493-CF1 and established the complete synthesis process. Through biological activity assays, we discovered that urs-monoamic acid is a synthetic intermediate of urs-monoamide and a potential plant growth inhibitor. In addition, we have developed various urbenonic acid derivatives, including the urbenyloxy derivative (UDA), which has high herbicidal activity without negatively affecting the growth of HeLa cells. We also found that urmotonic acid derivatives disrupt plant microtubules; in addition, KAND affects actin filaments and induces cell death; These multifaceted effects differ from those of known microtubule inhibitors and suggest a new mechanism of action for ursonic acid, which represents an important advantage in the development of new herbicides.
The discovery and practical application of beneficial natural products and their derivatives is a means of improving the quality of human life. Secondary metabolites produced by microorganisms, plants and insects have led to major advances in medicine and agriculture. Many antibiotics and anti-leukemia drugs have been developed from natural products. In addition, various types of pesticides, fungicides and herbicides are extracted from these natural products for use in agriculture. In particular, weed control herbicides are important tools for increasing crop yields in modern agriculture, and various types of compounds are already used commercially. Several cellular processes in plants, such as photosynthesis, amino acid metabolism, cell wall synthesis, regulation of mitosis, phytohormone signaling, or protein synthesis, are considered typical targets of herbicides. Compounds that inhibit microtubule function are a common class of herbicides that affect plant growth by affecting mitotic regulation2.
Microtubules are components of the cytoskeleton and are widely conserved in eukaryotic cells. The tubulin heterodimer consists of α-tubulin and β-tubulin forming linear microtubule protofilaments, with 13 protofilaments forming a cylindrical structure. Microtubules play multiple roles in plant cells, including determining cell shape, cell division, and intracellular transport3,4. Plant cells contain microtubules beneath the interphase plasma membrane, and these so-called cortical microtubules are thought to control the organization of cellulose microfibrils through the regulation of cellulose synthase complexes4,5. Cortical microtubules of root epidermal cells, present in the zone of rapid elongation of the root tip, are located laterally, and cellulose microfibers follow these microtubules and limit the direction of cell expansion, thereby promoting anisotropic cell elongation. Therefore, microtubule function is closely related to plant morphology. Amino acid substitutions in genes encoding tubulin cause skew of cortical microtubule arrays and left- or right-sided growth in Arabidopsis 6,7. Similarly, mutations in microtubule-associated proteins that regulate microtubule dynamics can also lead to distorted root growth8,9,10,11,12,13. In addition, treatment with microtubule-disrupting herbicides such as disopyramide, also known as pretilachlor, also causes left-sided oblique root growth14. These data indicate that precise regulation of microtubule function is critical for determining the direction of plant growth.
Various types of microtubule inhibitors have been discovered, and these drugs have made significant contributions to cytoskeletal research, as well as to agriculture and medicine2. In particular, oryzalin, dinitroaniline compounds, disopyramide, benzamide-related compounds, and their analogs can inhibit microtubule function and thereby inhibit plant growth. Therefore, they are widely used as herbicides. However, since microtubules are an important component of plant and animal cells, most microtubule inhibitors are cytotoxic to both cell types. Therefore, despite their recognized utility as herbicides, a limited number of antimicrotubule agents are used for practical purposes.
Streptomyces is a genus of the family Streptomyces, which includes aerobic, gram-positive, filamentous bacteria and is widely known for its ability to produce a wide range of secondary metabolites. Therefore, it is considered one of the most important sources of new biologically active natural products. In the current study, we discovered a new compound called coumamonamide, which was isolated from Streptomyces werraensis MK493-CF1 and S. werraensis ISP 5486. Using spectral analysis and full spectral analysis, the structure of coumamonamide was characterized and its unique N-alkoxypyrrole skeleton was determined. synthesis. Ursmonic acid, a synthetic intermediate of ursmonoamide and its derivatives, was found to inhibit the growth and germination of the popular model plant Arabidopsis thaliana. In a structure-activity relationship study, we found that a compound with C9 modified to ursonic acid, called nonyloxy derivative of ursonic acid (KAND), significantly enhances the inhibitory effect on growth and germination. Notably, the newly discovered plant growth inhibitor also affected the growth of tobacco and liverwort and was not cytotoxic to bacteria or HeLa cells. Moreover, some urmotonic acid derivatives induce a distorted root phenotype, implying that these derivatives directly or indirectly affect microtubules. Consistent with this idea, our observations of microtubules labeled either immunohistochemically or with fluorescent proteins indicate that KAND treatment depolymerizes microtubules. In addition, treatment with kumamotonic acid derivatives disrupted actin microfilaments. Thus, we have discovered a new plant growth inhibitor whose unique mechanism of action involves destruction of the cytoskeleton.
Strain MK493-CF1 was isolated from soil in Shinagawa-ku, Tokyo. Strain MK493-CF1 formed well-branched stromal mycelium. The partial sequence of the 16S ribosomal RNA gene (1422 bp) was determined. This strain is very similar to S. werraensis (NBRC 13404T = ISP 5486, 1421/1422 bp, T: typical strain, 99.93%). Based on this result, it was determined that this strain was closely related to the type strain of S. werraensis. Therefore, we provisionally named this strain S. werraensis MK493-CF1. S. werraensis ISP 5486T also produces the same bioactive compounds. Since there was little early research into obtaining natural products from this microorganism, further chemical research was carried out. After cultivation of S. werraensis MK493-CF1 on barley medium by solid-state fermentation at 30°C for 14 days, the medium was extracted with 50% EtOH. 60 ml of sample was dried to obtain 59.5 mg of crude extract. The crude extract was subjected to reverse phase HPLC to give N-methoxy-1H-pyrrole-2-carboxamide (1, named coumamonamide, 36.0 mg). The total amount of 1 is approximately 60% of the crude extract. Therefore, we decided to study in detail the properties of kumamotoamide 1.
Coumamonamide 1 is a white amorphous powder and high resolution mass spectrometry (HRESIMS) confirms C6H8N2O2 (Fig. 1). The C2-substituted pyrrole fragment of this compound is characterized by δH 6.94 (1H, t, J = 2.8, 4.8 Hz, H-4), δH 6.78 (1H, d, J = 2.5, δH in 1H NMR spectrum: 4.5 Hz, H-5) and δH 6.78 (1H, d, J = 2.5 Hz, H-6), and the 13C NMR spectrum shows the presence of four sp2 carbon atoms. The presence of an amide group at the C2 position was assessed by HMBC correlation from the C-3 proton to the amide carbonyl carbon at δC 161.1. In addition, 1 H and 13 C NMR peaks at δH 4.10 (3H, S) and δC 68.3 indicate the presence of N-methoxy groups in the molecule. Although the correct position of the methoxy group had not yet been determined using spectroscopic analysis such as enhanced difference spectroscopy and nuclear Overhauser abbreviation (NOEDF), N-methoxy-1H-pyrrole-2-carboxamide became the first candidate compound.
To determine the correct structure of 1, a total synthesis was performed (Fig. 2a). Treatment of commercially available 2-aminopyridine 2 with m-CPBA resulted in the corresponding N-oxide 3 in quantitative yield. After the 2-aminoazidation of 2, the cyclocondensation reaction described by Abramovich was carried out in benzene at 90°C to obtain the desired 1-hydroxy-1H-pyrrole-2-carbonitrile 5 in grams. Speed 60% (two stages). 15,16. Methylation and hydrolysis of 4 then gave 1-methoxy-1H-pyrrole-2-carboxylic acid (termed “cumotonic acid”, 6) in good yield (70%, two steps). Finally, amidation via acid chloride intermediate 6 using aqueous ammonia gave Kumamoto amide 1 in 98% yield. All spectral data of synthesized 1 were similar to isolated 1, so the structure of 1 was determined;
General synthesis and analysis of the biological activity of urbenamide and urbenic acid. (a) Total synthesis of Kumamoto amide. (b) Seven-day-old wild-type Arabidopsis Columbia (Col) seedlings were grown on Murashige and Skoog (MS) plates containing coumamonamide 6 or coumamonamide 1 at the indicated concentrations. Scale bar = 1 cm.
First, we assessed the biological activities of urbenamide and its intermediates for their ability to modulate plant growth. We added various concentrations of ursmonamide 1 or ursmonic acid 6 to MS agar medium and cultured Arabidopsis thaliana seedlings on this medium. These assays showed that high concentrations (500 μM) of 6 inhibited root growth (Fig. 2b). Next, we generated various derivatives by substituting the N1 position of 6 and performed structure–activity relationship studies on them (the analogue synthesis process is described in the Supporting Information (SI)). Arabidopsis seedlings were grown on a medium containing 50 μM ursonic acid derivatives, and root length was measured. as it shown on the picture. As shown in Figures 3a, b, and S1, coumamo acids have different lengths of linear alkoxy chains (9, 10, 11, 12, and 13) or large alkoxy chains (15, 16, and 17) at the N1 position. The derivatives showed significant inhibition of root growth. In addition, we found that application of 200 μM 10, 11, or 17 inhibited germination (Figs. 3c and S2).
Study of the structure-activity relationship of Kumamoto amide and related compounds. (a) Structure and synthesis scheme of analogues. (b) Quantification of root length of 7-day-old seedlings grown on MS medium with or without 50 μM coumamonamide derivatives. Asterisks indicate significant differences with sham treatment (t test, p < 0.05). n>18. Data are shown as mean ± SD. nt means “not tested” because more than 50% of the seeds did not germinate. (c) Quantification of germination rate of treated seeds incubated for 7 days in MS medium with or without 200 μM coumamonamide and related compounds. Asterisks indicate significant differences with sham treatment (chi-square test). n=96.
Interestingly, the addition of alkyl side chains longer than C9 reduced the inhibitory activity, suggesting that kumamotoic acid-related compounds require side chains of a certain size to exhibit their biological activity.
Because structure-activity relationship analysis showed that C9 was modified to ursonic acid and the nonyloxy derivative of ursonic acid (hereafter referred to as KAND 11) was the most effective plant growth inhibitor, we conducted a more detailed characterization of KAND 11. Treatment of Arabidopsis with 50 μM KAND 11 almost completely prevented germination, whereas lower concentrations (40, 30, 20, or 10 μM) of KAND 11 inhibited root growth in a dose-dependent manner (Fig. 4a, b). To test whether KAND 11 affects root meristem viability, we examined root meristems stained with propidium iodide (PI) and measured meristem area size. The size of the meristem of seedlings grown on a medium containing 25 μM KAND-11 was 151.1 ± 32.5 μm, while the size of the meristem of seedlings grown on a control medium containing DMSO was 264.7 ± 30.8 μm (Fig. 4c, d), which indicates that KAND-11 restores cellular activity. spreading. Root meristem. Consistent with this, KAND 11 treatment reduced the amount of cell division marker CDKB2;1p::CDKB2;1-GUS signal in the root meristem (Fig. 4e) 17 . These results indicate that KAND 11 inhibits root growth by reducing cell proliferation activity.
Analysis of the inhibitory effect of urbenonic acid derivatives (urbenyloxy derivatives) on growth. (a) 7-day-old wild-type Col seedlings grown on MS plates with the indicated concentrations of KAND 11. Scale bar = 1 cm. (b) Quantification of root length. Letters indicate significant differences (Tukey HSD test, p < 0.05). n>16. Data are shown as mean ± SD. (c) Confocal microscopy of propidium iodide-stained wild-type Col roots grown on MS plates with or without 25 μM KAND 11. White brackets indicate root meristem. Scale bar = 100 µm. (d) Quantification of root meristem size (n = 10 to 11). Statistical differences were determined using t-test (p < 0.05). The bars represent the average meristem size. (e) Differential interference contrast (DIC) microscopy of a root meristem containing the CDKB2 construct; 1pro: CDKB2; 1-GUS stained and stained on 5-day-old seedlings grown on MS plates with or without 25 µM KAND assay.
The phytotoxicity of KAND 11 was further tested using another dicotyledonous plant, tobacco (Nicotiana tabacum), and a major land plant model organism, liverwort (Marchantia polymorpha). As in the case of Arabidopsis, tobacco SR-1 seedlings grown on medium containing 25 μM KAND 11 produced shorter roots (Fig. 5a). Additionally, 40 of 48 seeds germinated on plates containing 200 μM KAND 11, whereas all 48 seeds germinated on mock-treated media, indicating that higher concentrations of KAND were significant (p < 0.05; chi test -square) inhibited the germination of tobacco. (Fig. 5b). In addition, the concentration of KAND 11 that inhibited bacterial growth in liverwort was similar to the effective concentration in Arabidopsis (Fig. 5c). These results indicate that KAND 11 can inhibit the growth of a variety of plants. We then investigated the possible cytotoxicity of bear monoamide-related compounds in other organisms, namely human HeLa cells and Escherichia coli strain DH5α, as representatives of higher animal and bacterial cells, respectively. In a series of cell proliferation assays, we observed that coumamonamide 1, coumamonamidic acid 6, and KAND 11 did not affect the growth of HeLa or E. coli cells at concentrations of 100 μM (Fig. 5d,e).
Growth inhibition of KAND 11 in non-Arabidopsis organisms. (a) Two-week-old wild-type SR-1 tobacco seedlings were grown on vertically positioned MS plates containing 25 μM KAND 11. (b) Two-week-old wild-type SR-1 tobacco seedlings were grown on horizontally positioned MS plates containing 200 μM KAND 11. ( c) Two-week-old wild-type Tak-1 liverwort buds grown on Gamborg B5 plates with the indicated concentrations of KAND 11. Red arrows indicate spores that stopped growing within the two-week incubation period. (d) Cell proliferation assay of HeLa cells. The number of viable cells was measured at fixed time intervals using a cell counting kit 8 (Dojindo). As a control, HeLa cells were treated with 5 μg/ml actinomycin D (Act D), which inhibits RNA polymerase transcription and causes cell death. Analyzes were performed in triplicate. (e) E. coli cell proliferation assay. E. coli growth was analyzed by measuring OD600. As a control, cells were treated with 50 μg/ml ampicillin (Amp), which inhibits bacterial cell wall synthesis. Analyzes were performed in triplicate.
To decipher the mechanism of action of cytotoxicity caused by uramide-related compounds, we reanalyzed urbenic acid derivatives with moderate inhibitory effects. as it shown on the picture. As shown in Figures 2b, 6a, seedlings grown on agar plates containing high concentrations (200 μM) of urmotonic acid 6 produced shorter and left-curved roots (θ = – 23.7 ± 6.1), whereas of seedlings grown on the control medium, the seedlings produced almost straight roots (θ = – 3.8 ± 7.1). This characteristic oblique growth is known to result from dysfunction of cortical microtubules14,18. Consistent with this finding, the microtubule-destabilizing drugs disopyramide and oryzalin induced similar root tilting under our growth conditions (Figs. 2b, 6a). At the same time, we tested urmotonic acid derivatives and selected several of them that, at certain concentrations, induced oblique root growth. Compounds 8, 9, and 15 changed the direction of root growth at 75 μM, 50 μM, and 40 μM, respectively, indicating that these compounds can effectively destabilize microtubules (Fig. 2b, 6a). We also tested the most potent ursolic acid derivative, KAND 11, at a lower concentration (15 µM) and found that application of KAND 11 inhibited root growth and that the direction of root growth was uneven, although they tended to slope to the left (Figure C3). . Because higher concentrations of microtubule-destabilizing drugs sometimes inhibit plant growth rather than cause root tilting, we subsequently assessed the possibility that KAND 11 affects microtubules by observing cortical microtubules in root epidermal cells. Immunohistochemistry using anti-β-tubulin antibodies in epidermal cells of seedling roots treated with 25 μM KAND 11 showed the disappearance of almost all cortical microtubules in epidermal cells in the elongation zone (Fig. 6b). These results indicate that kumamotonic acid and its derivatives act directly or indirectly on microtubules to disrupt them and that these compounds are novel microtubule inhibitors.
Ursonic acid and its derivatives alter cortical microtubules in Arabidopsis thaliana. (a) Root inclination angle measured in the presence of various urmotonic acid derivatives at the indicated concentrations. The effects of two compounds known to inhibit microtubules: disopyramide and oryzalin were also analyzed. The inset shows the standard used to measure root growth angle. Asterisks indicate significant differences with sham treatment (t test, p < 0.05). n>19. Scale bar = 1 cm. (b) Cortical microtubules in epidermal cells in the elongation zone. Microtubules in wild-type Arabidopsis Col roots grown on MS plates with or without 25 μM KAND 11 were visualized by immunohistochemical staining using β-tubulin primary antibodies and Alexa Fluor-conjugated secondary antibodies. Scale bar = 10 µm. (c) Mitotic structure of microtubules in the root meristem. Microtubules were visualized using immunohistochemical staining. Mitotic structures, including prophase zones, spindles, and phragmoplasts, were counted from confocal images. Arrows indicate mitotic microtubule structures. Asterisks indicate significant differences with sham treatment (t test, p < 0.05). n>9. Scale bar = 50 µm.
Although Ursa has the ability to disrupt microtubule function, its mechanism of action is expected to be different from typical microtubule depolymerizing agents. For example, higher concentrations of microtubule depolymerizing agents such as disopyramide and oryzalin induce anisotropic expansion of epidermal cells, whereas KAND 11 does not. In addition, co-application of KAND 11 and disopyramide resulted in a combined disopyramide-induced root growth response and KAND 11-induced growth inhibition was observed (Fig. S4). We also analyzed the response of the hypersensitive disopyramide 1-1 (phs1-1) mutant to KAND 11. phs1-1 has a non-canonical tubulin kinase point mutation and produces shorter roots when treated with disopyramide9,20. phs1-1 mutant seedlings grown on agar medium containing KAND 11 had shorter roots similar to those grown on disopyramid (fig. S5).
In addition, we observed mitotic microtubule structures, such as prophase zones, spindles, and phragmoplasts, in the root meristem of seedlings treated with KAND 11. Consistent with the observations for CDKB2;1p::CDKB2;1-GUS, a significant decrease in the number of mitotic microtubules was observed (Fig. .6c).
To characterize the cytotoxicity of KAND 11 at subcellular resolution, we treated tobacco BY-2 suspension cells with KAND 11 and observed their response. We first added KAND 11 to BY-2 cells expressing TagRFP-TUA6, which fluorescently labels microtubules, to assess the effect of KAND 11 on cortical microtubules. Cortical microtubule density was assessed using image analysis, which quantified the percentage of cytoskeletal pixels among cytoplasmic pixels. The assay results showed that after treatment with 50 μM or 100 μM KAND 11 for 1 hour, the density decreased significantly to 0.94 ± 0.74% or 0.23 ± 0.28%, respectively, while the density of cells treated with DMSO , amounted to 1.61 ± 0.34% (Fig. 7a). These results are consistent with the observation in Arabidopsis that KAND 11 treatment induces depolymerization of cortical microtubules (Fig. 6b). We also examined the BY-2 line with GFP-ABD-labeled actin filaments after treatment with the same concentration of KAND 11 and observed that KAND 11 treatment disrupted the actin filaments. Treatment with 50 μM or 100 μM KAND 11 for 1 h significantly reduced actin filament density to 1.20 ± 0.62% or 0.61 ± 0.26%, respectively, whereas the density in DMSO-treated cells was 1.69 ± 0.51% (Fig. 2). 7b). These results contrast with the effects of propyzamide, which does not affect actin filaments, and latrunculin B, an actin depolymerizer that does not affect microtubules ( SI Figure S6). In addition, treatment with coumamonamide 1, coumamonamide acid 6, or KAND 11 did not affect microtubules in HeLa cells (SI Figure S7). Thus, the mechanism of action of KAND 11 is believed to be different from that of known cytoskeleton disruptors. In addition, our microscopic observation of BY-2 cells treated with KAND 11 revealed the onset of cell death during KAND 11 treatment and showed that the proportion of Evans blue-stained dead cells did not increase significantly after 30 min of KAND 11 treatment, whereas after 90 minutes of treatment with 50 μM or 100 μM KAND, the number of dead cells increased to 43.7% or 80.1%, respectively (Fig. 7c). Taken together, these data indicate that the novel ursolic acid derivative KAND 11 is a plant-specific cytoskeletal inhibitor with a previously unknown mechanism of action.
KAND affects cortical microtubules, actin filaments, and viability of tobacco BY-2 cells. (a) Visualization of cortical microtubules in BY-2 cells in the presence of TagRFP-TUA6. BY-2 cells treated with KAND 11 (50 μM or 100 μM) or DMSO were examined by confocal microscopy. Cortical microtubule density was calculated from micrographs of 25 independent cells. Letters indicate significant differences (Tukey HSD test, p < 0.05). Scale bar = 10 µm. (b) Cortical actin filaments in BY-2 cells visualized in the presence of GFP-ABD2. BY-2 cells treated with KAND 11 (50 μM or 100 μM) or DMSO were examined by confocal microscopy. The density of cortical actin filaments was calculated from micrographs of 25 independent cells. Letters indicate significant differences (Tukey HSD test, p < 0.05). Scale bar = 10 µm. (c) Observation of dead BY-2 cells by Evans blue staining. BY-2 cells treated with KAND 11 (50 μM or 100 μM) or DMSO were examined by bright-field microscopy. n=3. Scale bar = 100 µm.
The discovery and application of new natural products has led to significant advances in various aspects of human life, including medicine and agriculture. Historical research has been carried out to obtain useful compounds from natural resources. In particular, actinomycetes are known to be useful as antiparasitic antibiotics for nematodes due to their ability to produce various secondary metabolites such as avermectin, the lead compound of ivermectin and bleomycin and its derivatives, used medicinally as an anticancer agent21,22. Likewise, a variety of herbicidal compounds have been discovered from actinomycetes, some of which are already used commercially1,23. Therefore, the analysis of actinomycete metabolites to isolate natural products with desired biological activities is considered an effective strategy. In this study, we discovered a new compound, coumamonamide, from S. werraensis and successfully synthesized it. Ursonic acid is a synthetic intermediate of urbenamide and its derivatives. It can cause characteristic root curling, exhibit moderate to strong herbicidal activity, and directly or indirectly damage plant microtubules. However, the mechanism of action of urmotonic acid may differ from that of existing microtubule inhibitors, since KAND 11 also disrupts actin filaments and causes cell death, suggesting a regulatory mechanism by which urmotonic acid and its derivatives influence a wide range of cytoskeletal structures. .
Further detailed characterization of urbenonic acid will help to better understand the mechanism of action of urbenonic acid. In particular, the next goal is to evaluate the ability of ursonic acid to bind to reduced microtubules to determine whether ursonic acid and its derivatives act directly on microtubules and depolymerize them, or whether their action results in microtubule destabilization. In addition, in the case where microtubules are not a direct target, identifying the site of action and molecular targets of ursonic acid on plant cells will help further understand the properties of related compounds and possible ways to improve herbicidal activity. Our bioactivity assay revealed the unique cytotoxic ability of ursonic acid on the growth of plants such as Arabidopsis thaliana, tobacco and liverwort, while neither E. coli nor HeLa cells were affected. Little or no toxicity to animal cells is an advantage of ursonic acid derivatives if they are developed as herbicides for use in open agricultural fields. Indeed, since microtubules are common structures in eukaryotes, their selective inhibition in plants is a key requirement for herbicides. For example, propyzamide, a microtubule depolymerizing agent that directly binds to tubulin and inhibits polymerization, is used as a herbicide due to its low toxicity to animal cells24. In contrast to disopyramide, related benzamides have different target specificities. In addition to plant microtubules, RH-4032 or benzoxamide also inhibits microtubules of animal cells or oomycetes, respectively, and zalilamide is used as a fungicide due to its low phytotoxicity25,26,27. The newly discovered bear and its derivatives exhibit selective cytotoxicity against plants, but it is worth noting that further modifications may alter their target specificity, potentially providing additional derivatives for the control of pathogenic fungi or oomycetes.
The unique properties of urbenonic acid and its derivatives are useful for their development as herbicides and use as research tools. The importance of the cytoskeleton in controlling plant cell shape is widely recognized. Earlier studies have shown that plants have evolved complex mechanisms of cortical microtubule organization by controlling microtubule dynamics to properly control morphogenesis. A large number of molecules responsible for the regulation of microtubule activity have been identified, and related research is still ongoing3,4,28. Our current understanding of microtubule dynamics in plant cells does not fully explain the mechanisms of cortical microtubule organization. For example, although both disopyramide and oryzalin can depolymerize microtubules, disopyramide causes severe root distortion while oryzalin has a relatively mild effect. Moreover, mutations in tubulin, which stabilizes microtubules, also cause dextrorotation in roots, whereas paclitaxel, which also stabilizes microtubule dynamics, does not. Therefore, studying and identifying the molecular targets of ursolic acid should provide new insights into the regulation of plant cortical microtubules. Likewise, future comparisons of chemicals that are effective in promoting distorted growth, such as disopyramide, and less effective chemicals, such as oryzalin or kumamotoric acid, will provide clues to how distorted growth occurs.
On the other hand, defense-related cytoskeletal rearrangements are another possibility to explain the cytotoxicity of ursonic acid. Infection of a pathogen or introduction of an elicitor into plant cells sometimes causes destruction of the cytoskeleton and subsequent cell death29. For example, oomycete-derived cryptoxanthin has been reported to disrupt microtubules and actin filaments prior to tobacco cell death, similar to what occurs with KAND treatment30,31. The similarities between defense responses and cellular responses induced by ursonic acid led us to hypothesize that they trigger common cellular processes, although a faster and stronger effect of ursonic acid than cryptoxanthin is evident. However, studies have shown that disruption of actin filaments promotes spontaneous cell death, which is not always accompanied by microtubule disruption29. In addition, it remains to be seen whether either the pathogen or elicitor causes distorted root growth, as ursonic acid derivatives do. Thus, molecular knowledge linking defense responses and the cytoskeleton is an attractive problem to be addressed. By exploiting the presence of low molecular weight compounds related to ursonic acid, as well as a range of derivatives with varying potencies, they may provide opportunities to target unknown cellular mechanisms.
Taken together, the discovery and application of new compounds that modulate microtubule dynamics will provide powerful methods to address the complex molecular mechanisms underlying plant cell shape determination. In this context, the recently developed compound urmotonic acid, which affects microtubules and actin filaments and induces cell death, may provide an opportunity to decipher the connection between microtubule control and these other mechanisms. Thus, chemical and biological analysis using urbenonic acid will help us understand the molecular regulatory mechanisms that control the plant cytoskeleton.
Inoculate S. werraensis MK493-CF1 into a 500 mL baffled Erlenmeyer flask containing 110 mL of seed medium consisting of 2% (w/v) galactose, 2% (w/v) Essence paste, 1% ( w/v) Bacto composition. -soyton (Thermo Fisher Scientific, Inc.), 0.5% (w/v) corn extract (KOGOSTCH Co., Ltd., Japan), 0.2% (w/v) (NH4)2SO4 and 0.2% CaCO3 in deionized water. (pH 7.4 before sterilization). The seed cultures were incubated on a rotary shaker (180 rpm) at 27°C for 2 days. Production cultivation via solid state fermentation. The seed culture (7 ml) was transferred into a 500 ml K-1 flask containing 40 g of production medium consisting of 15 g of pressed barley (MUSO Co., Ltd., Japan) and 25 g of deionized water (pH not adjusted before sterilization) . ). Fermentation was carried out at 30°C in the dark for 14 days. The fermentation material was extracted with 40 ml/bottle EtOH and centrifuged (1500 g, 4°C, 10 min). The culture supernatant (60 ml) was extracted with a mixture of 10% MeOH/EtOAc. The organic layer was evaporated under reduced pressure to obtain a residue (59.5 mg), which was subjected to HPLC with gradient elution (0–10 minutes: 90%) on a reverse phase column (SHISEIDO CAPCELL PAK C18 UG120, 5 μm, ID 10 mm × length 250 mm) H2O/CH3CN, 10–35 minutes: 90% H2O/CH3CN to 70% H2O/CH3CN (gradient), 35–45 minutes: 90% H2O/EtOH, 45–155 minutes: 90% H2O/EtOH to 100% EtOH (gradient (gradient), 155–200 min: 100% EtOH) at a flow rate of 1.5 ml/min, coumamonamide (1, 36.0 mg) was isolated as a white amorphous powder.
Kumamotoamide(1); 1H-NMR (500 MHz, CDCl3) δ 6.93 (t, J = 2.5 Hz, 1H), 6.76 (dd, J = 4.3, 1.8 Hz 1H), 6.05 (t , J = 3.8 Hz, 1H). ), 4.08 (s, 3H); 13C-NMR (125 MHz, CDCl3) δ 161.1, 121.0, 119.9, 112.2, 105.0, 68.3; ESI-HRMS [M+H]+: [C6H9N2O2]+ calculated value: 141.0659, measured value: 141.0663, IR νmax 3451, 3414, 3173, 2938, 1603, 1593, 1537 cm–1.
Columbia seeds (Col-0) were obtained from the Arabidopsis Biological Resource Center (ABRC) with permission for research use. Col-0 seeds were propagated and maintained under our laboratory conditions and used as wild-type Arabidopsis plants. Arabidopsis seeds were surface sterilized and cultured in half-strength Murashige and Skoog medium containing 2% sucrose (Fujifilm Wako Pure Chemical), 0.05% (w/v) 2-(4-morpholino)ethanesulfonic acid (MES) ( Fujifilm Wako Pure Chemical). ) and 1.5% agar (Fujifilm Wako Pure Chemical), pH 5.7, at 23 °C and constant light. Seeds of the phs1-1 mutant were provided by T. Hashimoto (Nara Institute of Science and Technology).
Seeds of strain SR-1 were provided by T. Hashimoto (Nara Institute of Science and Technology) and used as wild-type tobacco plants. Tobacco seeds were surface sterilized and soaked in sterile water for three nights to promote germination, then placed in a half-strength solution containing 2% sucrose, 0.05% (w/v) MES, and 0.8% gellan gum (Fujifilm Wako Pure Chemical) Murashige. and Skoog medium) with pH 5.7 and incubated at 23°C under constant light.
Strain Tak-1 was provided by T. Kohchi (Kyoto University) and was used as the standard experimental unit for the liverwort study. Gemma was obtained from sterilized cultured plants and then plated on Gamborg B5 medium (Fujifilm Wako Pure Chemical) containing 1% sucrose and 0.3% gellan gum and incubated at 23°C under continuous light.
Tobacco BY-2 cells (Nicotiana tabacum L. cv. Bright Yellow 2) were provided by S. Hasezawa (University of Tokyo). BY-2 cells were diluted 95-fold in modified Linsmeier and Skoog medium and supplemented weekly with 2,4-dichlorophenoxyacetic acid 32 . The cell suspension was mixed on a rotary shaker at 130 rpm at 27°C in the dark. Wash cells with 10 times the volume of fresh medium and resuspend in the same medium. BY-2 transgenic cell lines stably expressing the microtubule marker TagRFP-TUA6 or the actin filament marker GFP-ABD2 under the cauliflower mosaic virus 35S promoter were generated as described33,34,35. These cell lines can be maintained and synchronized using procedures similar to those used for the original BY-2 cell line.
HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies) supplemented with 10% fetal bovine serum, 1.2 U/ml penicillin, and 1.2 μg/ml streptomycin in a 37°C incubator with 5% CO2.
All experiments described in this manuscript were performed in accordance with Japanese biosafety regulations and guidelines.
Compounds were dissolved in dimethyl sulfoxide (DMSO; Fujifilm Wako Pure Chemical) as stock solutions and diluted in MS medium for Arabidopsis and tobacco or Gamborg B5 medium for liverwort. For the root growth inhibition assay, more than 10 seeds per plate were sown on agar medium containing the indicated compounds or DMSO. Seeds were incubated in a growth chamber for 7 days. The seedlings were photographed and the length of the roots was measured. For Arabidopsis germination assay, 48 seeds per plate were sown on agar medium containing 200 μM compound or DMSO. Arabidopsis seeds were grown in a growth chamber and the number of germinated seedlings was counted 7 days after germination (dag). For tobacco germination assay, 24 seeds per plate were sown on agar medium containing 200 μM KAND or DMSO. Tobacco seeds were grown in a growth chamber and the number of germinated seedlings was counted after 14 days. For the liverwort growth inhibition assay, 9 embryos from each plate were plated on agar medium containing the indicated concentrations of KAND or DMSO and incubated in a growth chamber for 14 days.
Use seedlings stained with 5 mg/ml propidium iodide (PI) to visualize root meristem organization. PI signals were observed by fluorescence microscopy using a TCS SPE confocal laser scanning microscope (Leica Microsystems).
Histochemical staining of roots with β-glucuronidase (GUS) was performed according to the protocol described by Malami and Benfey36. Seedlings were fixed in 90% acetone overnight, stained with 0.5 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid in GUS buffer for 1 hour and placed in a hydrated chloraldehyde solution. (8 g chloral hydrate, 2 ml water and 1 ml glycerol) and observed by differential interference contrast microscopy using an Axio Imager M1 microscope (Carl Zeiss).
Root angles were measured on 7-day-old seedlings grown on vertically placed plates. Measure the angle of the root from the direction of the gravity vector as described in step 6.
The arrangement of cortical microtubules was observed as described, with minor modifications to the protocol 37 . Anti-β-tubulin antibody (KMX-1, Merk Millipore: MAB3408) and Alexa Fluor 488-conjugated anti-mouse IgG (Thermo Fisher Scientific: A32723) were used as primary and secondary antibodies at 1:1000 and 1:100 dilutions, respectively. Fluorescence images were acquired using a TCS SPE confocal laser scanning microscope (Leica Microsystems). Acquire Z-stack images and create maximum intensity projections according to the manufacturer’s instructions.
HeLa cell proliferation assay was performed using Cell Counting Kit 8 (Dojindo) according to the manufacturer’s instructions.
The growth of E. coli DH5α was analyzed by measuring cell density in culture using a spectrophotometer at 600 nm (OD600).
Cytoskeletal organization in transgenic BY-2 cells was observed using a fluorescence microscope equipped with a CSU-X1 confocal scanning device (Yokogawa) and an sCMOS camera (Zyla, Andor Technology). Cytoskeletal density was assessed by image analysis, which quantified the percentage of cytoskeletal pixels among cytoplasmic pixels in confocal images using ImageJ software as described38,39.
To detect cell death in BY-2 cells, an aliquot of the cell suspension was incubated with 0.05% Evans blue for 10 minutes at room temperature. Selective Evans blue staining of dead cells depends on extrusion of the dye from viable cells by the intact plasma membrane40. Stained cells were observed using a bright-field microscope (BX53, Olympus).
HeLa cells were grown in DMEM supplemented with 10% FBS in a humidified incubator at 37°C and 5% CO2. Cells were treated with 100 μM KAND 11, kumamonamic acid 6, kumamonamide 1, 100 ng/ml colcemid (Gibco), or 100 ng/ml Nocodmaze (Sigma) for 6 h at 37°C. Cells were fixed with MetOH for 10 min and then with acetate for 5 min at room temperature. Fixed cells were incubated with β-tubulin primary antibody (1D4A4, Proteintech: 66240-1) diluted in 0.5% BSA/PBS for 2 hours, washed 3 times with TBST, and then incubated with Alexa Fluor goat antibody. 488 1 hour. – Mouse IgG (Thermo Fisher Scientific: A11001) and 15 ng/ml 4′,6-diamidino-2-phenylindole (DAPI) diluted in 0.5% BSA/PBS. After washing with TBST three times, stained cells were observed on a Nikon Eclipse Ti-E inverted microscope. Images were captured with a cooled Hamamatsu ORCA-R2 CCD camera using MetaMorph software (Molecular Devices).
Post time: Jun-17-2024