Sea sand

Fresh plant material was ground with sea sand (p.a.; Merck, Darmstadt, Germany) using a mortar and pestle in 50 mM MES buffer, pH 6.0 (100 µL buffer per 100 mg plant material). I3CN (25 or 20 µL of 1 nmol µL⁻¹ in MeOH) and benzonitrile (30 or 25 µL of 100 ng µL⁻¹ in MeOH) were added as internal standards. After 10 min incubation at room temperature, plant parts were removed by centrifugation, and the aqueous solution was extracted twice with 750 µL dichloromethane. Organic phases were passed over Na₂SO₄, dried in an air stream to complete dryness, dissolved in 75 µL MSTFA/pyridine (2 + 1), incubated at 70 °C for 1 h and analyzed by GC-MS. Indolic breakdown products were identified by comparison of mass spectra and retention times with those of the standard mixture. Quantification was achieved based on peak areas determined from the RIC of the molecular ions using the experimentally determined response factors (Supplementary Material Table S2).

Each bacterial strain was cultured separately in 1 L of TSB at 28°C for 48 h. The cultured broth was centrifuged at 10,000 × g for 20 min at 4°C to obtain supernatant which was then subjected to fractionation. Briefly, the same volume (1 L) of hexane was mixed with the supernatant to separate organic and aqueous fractions. The resulting aqueous fraction was combined with the same volume of ethyl acetate. These processes were sequentially repeated for chloroform and butanol organic solvents. Resulting organic extracts [hexane extract (HEX), ethyl acetate extract (EAX), chloroform extract (CX), and butanol extract (BX)] containing bacterial metabolites were dried with a rotary evaporator (Eyela N-1110, Rikakikai, Tokyo, Japan) at 20°C for HEX, 25°C for CX, 30°C for EAX, and 40°C for BX. After weighing dried metabolites, each extract was resuspended with 5 mL of methanol. Resulting metabolites were subjected to TLC using silica gel plates (20 cm × 20 cm; Merck, Darmstadt, Germany). After developing with chloroform:methanol:acetic acid (7:2.5:0.5, v/v) as an eluent, silica gel plates were incubated with a mixture (19:1, g/g) of sea sand (Merck) and iodine (Duksan, Ansan, Korea). Spots were then visualized in a fluorescence analysis cabinet (Spectroline, CM-10, Westbury, NY, United States).

X. hominickii was cultured in 1 L of TSB at 28°C for 48 h. After centrifuging cultured broth at 10,000 × g for 20 min at 4°C, the resulting supernatant was used for organic extraction as described by Mollah et al. [48 (link)]. Briefly, the supernatant was mixed with the same volume of hexane. After 30 min of incubation 4°C, the hexane extract (HEX) was separated from the aqueous fraction. The same procedure was sequentially used to obtain chloroform (CX), ethylacetate (EAX), and butanol (BX) extracts. Resulting organic extracts containing bacterial metabolites were dried with a rotary evaporator (Eyela N-1110, Rikakikai, Tokyo, Japan). After weighing, extracts were resuspended in methanol. TLC was performed for resulting extracts to obtain metabolites on a silica gel plate (20×20 cm; Merck, Darmstadt, Germany). Different compositions of chloroform and methanol (v/v) were used as eluents. The developed silica gel plate was incubated with a mixture (19:1, g/g) of sea sand (Merck) and iodine (Duksan, Ansan, Korea). Spots were visualized and marked in a fluorescence analysis cabinet (Spectroline, CM-10, Westbury, NY, USA).

The porous material used was the commercially available Merck purified sea sand with an average particle size of 100–300 µm (product no. 1.07711.5000). The sand was already purified by the manufacturer with acidic treatment and calcination. In another treatment step the sea sand was put in MilliQ water with a volume to mass ratio of 2:1. A small aliquot of 100 µL HNO₃ was added to the suspension (pH ~3–4), which was then vigorously agitated for a few seconds. The turbid supernatant was poured out of the vessel a few seconds after agitation, giving enough time for the biggest particles to settle leaving the fines in suspension. This procedure was repeated 7-10 times until the supernatant appeared clear and a stable and a neutral pH was reached. The sand was then dried in an oven at 105 °C for at least 24 h (this sample is later referred as cleaned sand; Table 3). The N₂-BET surface area of the powder was equal to 0.55 ± 0.02 m² g⁻¹. The mineralogical composition of the sand powder was characterized via XRD and XRF. The porosity and petro-physical properties of the compacted sand were determined with mercury intrusion porosimetry (MIP, before the formation of a precipitation front) and µ-CT (before and after the formation of a precipitation front).

For each sample, pollen grains were isolated from anthers collected from 60 flowers, at developmental stages A-4 and A-3, as detailed above for protein extraction. The released pollen grains were immediately frozen in liquid nitrogen and kept frozen at -80^oC until use. Pollen grains were ground to a fine powder using liquid nitrogen and sea sand (Merck, Darmstadt, Germany) and total RNA was extracted using the Tri reagent (Sigma-Aldrich, Israel). RNA was treated with TURBO DNase (AB Applied Biosystems, Ambion, CA, United States) according to the manufacturer’s instructions. Two micrograms of total RNA were used for cDNA synthesis, using Maxima first-strand cDNA synthesis kit (Thermo Scientific³) according to the manufacturer’s instructions. Each qPCR reaction was performed with three biological replicates, each with three technical replicates. The PCRs were performed with Platinum SYBR Green qPCR Super Mix-UDG (Invitrogen, Germany) in a Rotor-Gene 6000 cycler (Qiagen, Germany). PCR products were analyzed using Rotor Gene Series 6000 software version 1.7 (Qiagen, Germany). Values in each sample were normalized to the level of 18S ribosomal gene as a reference gene (Frank et al., 2009 (link)). All primers used are listed in Supplementary Table S13.

Okra (mature and ready to be consumed) was purchased from local supermarkets in 2019 when the study is conducted. Azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), ascorbic acid, Acarbose, HPLC grade water, sea sand and formic acid were purchased from Sigma-Aldrich (Singapore). All cell culture reagents, including trypsin, MCDB-131 media, Fetal Bovine Serum (FBS), Attachment Factor (AF), 0.4% trypan blue and L-glutamine acid were purchased from Thermofisher Scientific (Singapore). All other reagents used throughout the experiments in this study were of standard analytical grade.
The whole okra plants were washed and dried before they were separated into various parts, consisting of the Seeds, Outer Skin and Inner Skin. Subsequently, the various okra parts were oven-dried at 40 °C for 24 h [23 (link)]. The samples were crushed using mortar and pestle after being cooled down to generate a more homogeneous and representative sample. Samples were sieved individually to separate into following particle sizes: below 0.3 mm, between 0.3 mm and 2.8 mm, and above 2.8 mm. Fine samples with particle size below 0.3 mm was used for chemical analysis.

Extractions of algae species were performed using an accelerated solvent extractor (ASE, 350, Dionex Corp, Sunnyvale, CA, USA) equipped with a solvent controlled unit. Five different solvents (hexane, ethanol, ethyl acetate, acetone and ethanol 50%) at 3 different temperatures (80 °C, 120 °C, 160 °C) were tested. Extractions were performed in triplicate in 10 mL extractions cells and 100 bar. The amount of 1 g of algae was loaded into the stainless steel cell with sea sand (thin grain, particle size 250–300 μm, Sigma-Aldrich, Madrid, Spain) above and below the sample to avoid any void spaces. Then, the extraction cell was placed into the carrousel and the automatic extraction sequence began with the loading of the cell into the oven. When the cell was heated to the pre-set extraction temperature, the cell was pressurized for 10 min and then allowed to flow the extract into the collection vial. The solvent total volume used was 20 mL.