Syntheses of Hydroxylated BOA Species
Except for BOA-6-OH, hydroxylated benzoxazolinones are commercially not available. Therefore, the isomers of hydroxylated benzoxazolinones, including BOA-6-OH, were synthesized to compare their detoxification in maize seedlings and to study gene expression. However, the laborious syntheses and yields of the different isomers did not allow to perform all experiments with all isomers.
2-Nitroresorcinol (1) was prepared by nitration of resorcinol with conc. H2SO4 and 67% HNO3. 1 was isolated by steam distillation as orange crystals (24% yield, 11 g). The melting points (mp) of 85°C was in accordance with Schaffrath (1970). NMR spectra confirmed the structure: 1H-NMR (CDCl3, 300 MHz) δ 10.65 (2 H, s, O-H), 7.44 (1 H, t, J = 8.4 Hz, H-5), 6.61 (2 H, d, J = 8.4 Hz, H-4, H-6). 13C-NMR (CDCl3, 75 MHz) δ 156.5 (C, C-1, C‑3). 138.9 (CH, C-5), 123.9 (C, C-2), 109.6 (CH, C-4, C-6).
2-Nitrohydroquinone (2) was prepared by the classical Elbs oxidation of 2-nitrophenol with K2S2O8 in diluted NaOH solution (Elbs 1893). 2 was obtained as dark red crystals in 9 % yield and 3 g amount. The purification was done by removal of unreacted 2-nitrophenol by steam distillation followed by extraction of 2 from the residue with MTBE (methyl t-butyl ether) and a final crystallization of the MTBE extract from water. The mp of 131-132 °C is in accordance with the data of Elbs (1893). 1H-NMR (DMSO-d6, 300 MHz) δ10.14 (1 H, brs, O-H), 9.60 (1 H, brs, O-H), 7.22 (1 H, d, J = 2.9 Hz, H-3),7.02 (1H, dd, J = 9.0, 2.9 Hz, H-5), 6.96 (1 H, d, J = 9.0 Hz, H-6). 13C-NMR (DMSO-d6, 75 MHz) δ149.7 (C, C-4), 145.1 (C, C-1), 135.8 (C, C-2), 124.3 (CH, C-5), 120.1 (CH, C-6), 109.5 (CH, C-3).
4-Nitroresorcinol (3) was prepared from resorcinol according to the recent nitration method of Samajdar et al. (2000) with silica gel-supported Bi(NO3)3 x 5 H2O with some variations (silica gel instead of montmorillonite, no microwave irradiation, CH2Cl2 as solvent during the reaction with the supported reagent). 3 was isolated by extraction with CHCl3 and purified by flash chromatography over silica gel (eluent n-hexane/ethyl acetate 2:1 v/v) as yellow crystals in 26% yield and 2 g amount. The mp of 120 °C is in accordance with Kauffmann and Kugel (1911). 1H-NMR (DMSO-d6, 300 MHz) δ 10.95 (1 H, brs, O-H), 10.80 (1 H, brs, O-H), 7.88 (1 H, d, J = 9.1 Hz, H-5), 6.42 (1H, d, J = 2.4 Hz, H-6), 6.39 (1 H, dd, J = 9.1, 2.4 Hz, H-2). 13C-NMR (DMSO-d6, 75 MHz) δ 164.9 (C, C-1), 156.0 (C, C-3), 128.1 (C, C-4), 127.8 (CH, C-5), 108.8 (CH, C-6), 103.6 (CH, C-2).
3-Nitrocatechol (4) was prepared by nitration of an ethereal solution of catechol with 100% fuming nitric acid according to Rosenblatt et al. (1953). 3 was separated from the accompanying 4-nitrocatechol by steam distillation as dark yellow crystals in 15% yield and 4 g amount. The mp of 87 °C was in accordance with the literature (Samajdar et al. 2000). 1H-NMR (CDCl3, 300 MHz) δ10.62 (1 H, brs, O-H), 7.65 (1 H, dd, J = 8.7, 1.5 Hz, H-4), 7.24 (1 H, dd, J = 8.4, 1.2 Hz, H-6), 6.91 (1H, dd, J = 8.7, 8.4 Hz, H-5), 5.82 (1 H, brs, O-H). 13C-NMR (CDCl3, 75 MHz) δ 146.5 (C, C-1), 142.8 (C, C-2), 133.8 (C, C-3), 121.7 (CH, C-5), 119.8 (CH, C-6), 115.8 (CH, C-4).
General Method for the Synthesis of the Hydroxy-Substituted BOAs 5-8.
In a glass hydrogenation flask, 1.54 g (9.9 mmol) of the corresponding nitro compound (compounds 1-4) were dissolved in 150 ml of dry THF followed by 0.5 g Pd/C catalyst. The mixture was stirred under hydrogen from a balloon at standard pressure for 6 h. The H2 balloon was then removed, the flask was cooled in an ice water bath and 2.3 g (22 mol; 3.0 ml) triethylamine was added at once followed by dropwise addition of 0.8 g (2.6 mmol) triphosgene dissolved in 50 ml dry THF over a dropping funnel within 3 min. The mixture was stirred for 30 min. Then, the catalyst and the triethylamine hydrochloride were filtered. The solvent was removed from the filtrate in vacuo. The remaining residue was purified by column chromatography using CH2Cl2/methanol 10:1 as the eluent. An exception is compound 7. All melting points given here were in accordance with those reported by Zinner and Wigert (1960), who used a different urea-based synthesis. The structures were further confirmed by 1H- and 13C-NMR, IR and MS. NMR analyses were done with DRX-400 and Fourier 300 instruments (Bruker). The IR-spectra were obtained with a Fourier Transform Infrared Spectrometer FT/IR-4100 (JASCO). The EI-MS data were acquired on a MAT 8230 spectrometer (Thermo Fisher, formerly Finnigan MAT). Melting points were determined with a Boëtius micro hot stage.
4-Hydroxybenzoxazolin-2(3H)-one 5: Yield: 998 mg (66 %) off-white crystals, mp. 288-289°C (Zinner and Wigert 1960: 281-283°C). 1H-NMR (DMSO-d6, 300 MHz) δ10.69 (2 H, brs, O-H, N-H), 6.86 (1 H, t, J = 8.2 Hz, H-6), 6.71 (1 H, d, J = 8.2 Hz, H-7), 6.63 (1 H, d, J = 8.2 Hz, H-5). 13C-NMR (DMSO-d6, 75 MHz) δ 154.5 (C, C-2), 144.7 (C, C-7a), 142.0 (C, C-4), 122.0 (CH, C-6), 118.2 (C, C-3a), 110.9 (CH, C-5), 100.9 (CH, C-7). IR (KBr): = 3449, 3287, 1738, 1476, 1024. EI-MS m/z (%): 151(M+, 100), 79 (29), 67 (30), 57 (60), 43 (39).
5-Hydroxybenzoxazolin-2(3H)-one 6: Yield: 651 mg (43 %) beige crystals, mp. 212-213°C (Zinner and Wigert 1960: 209-210°C). 1H-NMR (DMSO-d6, 300 MHz) δ 11.33 (1 H, brs, N-H), 9.36 (1 H, brs, O-H), 7.02 (1 H, d, J = 8.6 Hz, H-7), 6.47 (1H, d, J = 2.4 Hz, H-4), 6.41 (1 H, dd, J = 8.6, 2.4 Hz, H-6). 13C-NMR (DMSO-d6, 75 MHz) δ155.1 (C, C-2), 154.1 (C, C-5), 136.3 (C, C-7a), 131.0 (C, C-3a), 109.9 (CH, C-7), 108.0 (CH, C-6), 97.4 (CH, C-4).IR (KBr): = 3541, 3464, 3285, 1731, 1482, 1172. EI-MS m/z (%): 151(M+, 100), 95 (95), 68 (36), 67 (32), 41 (29).
6-Hydroxybenzoxazolin-2(3H)-one 7: A black solid remained after removal of the solvent in vacuo. It was refluxed for 10 min along with 0.5 g activated charcoal powder and 200 ml of distilled water. It was then filtered and cooled to yield 250 mg (17%) of 7 as colorless crystals, mp 299 °C (Zinner and Wigert 1960: 288-292 °C). Due to the high mp, the solubility of 7 is poor and column chromatography was not advisable. 1H-NMR (DMSO-d6, 300 MHz) δ 11.24 (1 H, brs, N-H), 9.36 (1 H, brs, O-H), 6.86 (1 H, d, J = 8.5 Hz, H-4), 6.69 (1H, d, J = 2.2 Hz, H-7), 6.55 (1 H, dd, J = 8.5, 2.2 Hz, H-5). 13C-NMR (DMSO-d6, 75 MHz) δ154.8 (C, C-2), 153.1 (C, C-6), 144.1 (C, C-7a), 122.3 (C, C-3a), 110.1 (CH, C-5), 109.9 (CH, C-4), 98.0 (CH, C-7). IR (KBr): = 3458, 3214, 1734, 1499, 1106. EI-MS m/z (%): 151(M+, 100), 95 (87), 67 (39), 52 (46), 41 (31).
7-Hydroxybenzoxazolin-2(3H)-one 8: Yield: 420 mg (29 %) beige crystals, mp 236-238 °C (Zinner and Wigert, 1960: 232 °C). 1H-NMR (DMSO-d6, 300 MHz) δ 10.50 (2 H, brs, N-H, O-H), 6.90 (1H, dd, J = 8.4, 7.8 Hz, H-5), 6.56 (1 H, dd, J = 8.4, 1.1 Hz, H-4), 6.51 (1 H, dd, J = 7.8, 1.1 Hz, H-6). 13C-NMR (DMSO-d6, 75 MHz) δ 154.5 (C, C-2), 140.9 (C, C-7), 131.8 (C, C-3a), 131.1 (C, C-7a), 124.2 (CH, C-5), 110.3 (CH, C-4), 110.9 (CH, C-6).
IR (KBr): = 3360, 3261, 1768, 1383, 1150. EI-MS m/z (%): 151(M+, 100), 96 (47), 68 (36), 67 (40), 52 (33).
Plant material
Caryopses of Zea mays cultivar Cassila (KWS, Germany) were hydroponically grown for 7 days as described in Schulz et al. (2016). A. theophrasti was grown as described in Schulz et al. (2017).
For the analysis of detoxification products, the maize seedlings were incubated with the BOA-isomers for 24h (3 seedlings/ 20 ml of 0.5 mM of one of the isomers in tap water). For analysis of BOA-6-O-glc accumulation during short term exposure, seedlings were incubated 0, 10, 20, 30, 40, 50 and 60 min with 0.5 µM BOA-6-OH. Analysis and determination of the detoxification products was performed by HPLC using the system and the method described below. For preparation of 500 ml incubation solution the isomers were pre-solved in 1 ml methanol and sonicated for 5 min before adding tap water. Incubations with 9 seedlings/60 ml were repeated at least 6 times for each BOA-OH isomer. Browning reactions were monitored after 30min, 1h, 6h and 24h, bubble formation after 10 min, 30 min, 1 h and 6h. Root extracts were performed as described in Schulz and Wieland (1998). Peroxidase activity at living root surfaces was detected by bathing the roots (1 seedling / 15 ml Falcon tube) in 10 ml 0.1 mM acetate buffer pH 5.0 supplemented with 1.6 ml 2 mM ABTS (Roche Diagnostics, Germany) and 0.6 ml 15 mM H2O2 (Sigma-Merck, Germany). Sites with peroxidase activity immediately turned dark or black-green. Catalase activity elicited by BOA-6,5,4-OH was concluded from bubble formation according to Schellhorn and Stones (1992) and Wheelis (2008), presenting a non-destructive method.
Benzoxazinoids in Root Hairs and their Wash Solutions
For the harvest of root hairs, maize was germinated on wet filter paper placed in Petri dishes. Approximately 1 mg root hairs from six 7-day-old maize seedlings were cut and immediately collected in a cap with 500 µl 70% ice cold methanol (n=12). From another six seedlings, root hair zones were washed with 500 µl water and the wash solutions including the mucilage drops (see below) collected in pure methanol, yielding a 50% solution. Four wash solutions collected over a period of 4 h were combined for one sample. Root hair extracts and wash solutions were analyzed by HPLC/ DAD (Shimadzu, Germany), equipped with a RP-18 column (Macherey-Nagel, Düren, EC 250/4.6 Nucleodur 100-5 C18). Linear gradients were run with 0.01% formic acid/H2O and methanol within 35 min. For confirmation of the compounds identity, root hair extracts were subsequently analyzed by Ultra Performance Liquid Chromatography (UPLC)-electrospray (ESI)-mass spectrometry (Waters, Eschborn) as described in Schulz et al. (2016). Mucilage drops from the root tips of 6 seedlings were collected on ice with a syringe and analyzed by HPLC. Since only traces of the BXs were found, further wash solutions contained washes of root hair zone including the mucilage drops (n=7).
Lipid Analyses
For total fatty acids methyl ester (FAME) determinations, 35 root tips (0-2cm, further named ZmRTa) /sample and 10 older root parts (2-4 cm, further named ZmRTb)/sample from maize and 13 root tips (AbRTa)/ sample and 7 older root parts (AbRTb) /sample from A. theophrasti were harvested without incubation and after 10, 20, 30, 40, 50 and 60 min incubations in 0.5 mM BOA-6-OH. For these experiments, BOA-6-OH was purchased from Merck-Sigma. The harvested root material from each incubation time was immediately treated with boiling water for 20 min, followed by 3x repeated chloroform/methanol (1:2), (1 ml/sample) extractions. The resulting three portions of crude lipid extracts were combined and purified by addition of 0.75 ml 0.3 M ammonium acetate. After centrifugation at 2000 g for 5 min, the lipid phase was dried under N2. When not directly analyzed, the extracts were stored at -20 °C until analysis. The extracted root material was dried overnight at 104 °C for dry weight determination. The described extraction method followed the procedure described by Siebers et al. (2018). The conversion of fatty acids into fatty acids methyl esters (FAMEs) was done according to Browse et al. (1986). For quantification pentadecanoic acid, (15:0) was added as internal standard to the samples. FAMEs were analyzed using an Agilent 7890 gas chromatograph with Supelco SP-2380 capillary column and a flame ionization detector (7890 Gas chromatograph (GC) with flame ionization detector (FID) Agilent, Böblingen (D) as described in Siebers et al. (2018). The oven temperature was 100 °C initially, increased to 160 °C at 25 °C min-1, and finally ramped to 220 °C at 10 °C min-1. Fatty acid peaks were identified using FAME standards (Sigma-Aldrich, Supelco® 37 Component FAME Mix).
For the measurement of phospholipids, glycolipids, phospholipid fatty acid (PLFA) analyses and determinations of TAGs and DAGs, 4 maize root parts/sample (see above) or 7 root parts/sample of A. theophrasti were extracted as described. Lipid extraction and analysis were according to Bravo et al. (2017). Lipids were fractionated according to their polarity by solid phase extraction (Gasulla et al. 2013; Siebers et al. 2018; Schütz et al. 2021). The fractionated phospholipids were methanolized and converted into their methyl esters prior to measurement by GC (Browse et al. 1986). The contents and molecular species composition of phospholipids/glycolipids, TAGs and DAGs were determined by direct infusion mass spectrometry on an Agilent Accurate Mass quadrupole time-of-flight (Q-TOF) mass spectrometer. Quantification of lipid molecular species was performed by MS/MS analysis.
Gene Expression Studies
Choice of Genes
The selection of genes for comparative expression studies was restricted to SOD2, CAT 1, CAT 3, FAD2-1, FAD2-2, PR1, PR4 and POX 12 because of the limited amounts of BOA-4, 5- and 7-OH available for the experiments. For the same reason, the time points for real- time PCR were restricted, but within the most reactive period during the incubation regarding early gene responses, starting polymerization and bubble development. The set of genes was selected to give first clues on the oxidative stress elicitation by BOA-OH isomers, lipid repair and possible relations to plant immune responses in the youngest root tissues, thought as a preliminary base for future in depth investigations. Superoxide dismutase 2 (SOD2) involved in superoxide radical transfer to H2O2 was included as an indicator for ROS stress (Gond et al. 2015). Mitochondrial catalase (CAT3) gene is responsive to H2O2, reactive oxygen species and xenobiotics. Transcripts were detected in the root, epicotyl, and leaves (Redinbaugh et al. 1990). CAT1 was described to be expressed in roots, shoot, and scutellum of young seedlings (Scandalios et al. 1983). While the CAT1 expression is responsive to oxidative stress, the protein is localized to the cytosol (Mylona et al. 2007). CAT 2 expression was described to be light responsive and was therefore not included in the studies (Guan et al. 1996). Expression of members of the complex gene families with functions in the glutathione–ascorbate (GSH–ASC) cycle, such as ascorbate peroxidase producing H2O from H2O2, could not be addressed in this study. The gene expression of two ER-bound oleoyl desaturases (FAD2-1 and FAD2-2) was studied in context with lipid restoration processes after BOA-6-OH exposure. The enzymes synthesize linoleic acid (18:2) mostly in non-photosynthetic tissue (Dar et al. 2017). The seed and bud specific desaturase FAD2-1 was considered not to be responsive, but effects of allelochemicals on FAD2-1 expression are not known. FAD2-2 was described as lowly expressed during the entire life cycle of the plant. To study whether the BOA-OHs induce the expression of genes related to pathogenesis and resistance, induction of POX 12, PR1, PR4 and NPR1 expression was studied (Sajad et al. 2018; Backer et al. 2019; Hemetsberger et al. 2012). Primers are given in Tab. S 1.
RNA Isolation, cDNA Synthesis and qRT-PCR
7-day-old maize seedlings were incubated with 0.5 mM of the BOA-OH isomers for 30 min, 1 h and 6 h, for SOD2 expression, BOA-6/5-OH samples in addition for 24 h, for FAD2-2 expression all isomers also for 24 h. 100 mg Root tips were cut (3 cm) and immediately frozen in liquid nitrogen until RNA was extracted. The material was homogenized with the Precellys® 24 / 24-Dual (PEQLAB Biotechnologie, Erlangen) and RNA was isolated with the NucleoSpin RNA Plant Kit (Macherey and Nagel, Düren) according the instructions of the manufacturer. For cDNA preparation the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit was used. RT-PCR was performed with a 7300 real time PCR system (Applied Biosystems, Darmstadt). The reaction mixture (20 µl) consisted of 4 µl H2O, 5 µl cDNA, 1 µl primer pair (working solution 10 pmol/µl) and 10 µl 5xEvaGreen (ROX)qPCR-Mix II (Bio-Budget Technologies, Krefeld, Germany). Gene expression was determined relative to the control plants and to ZmActin as reference gene. For evaluation of RT-qPCR results, the ΔΔct method was applied.
Statistics
Bars, presented with SD, were established with data obtained from three biological replicates unless otherwise noted. Statistical analysis of all the data was done with the t-test. Data in the figures are presented as the mean ± standard deviation. Significant differences between samples and the controls are indicated: ∗p < 0.05; ∗∗p < 0.005; ∗∗∗p < 0.0005.