Isoprenoids

About 50 g of the sinter samples were Soxhlet extracted (24 h) with a mixture (ca. 250 ml) of dichloromethane/methanol (DCM/MeOH, 3:1, v/v), after addition of internal standards (tetracosane-D₅₀, myristic acid-D₂₇, 2-hexadecanol). The total lipid extracts were concentrated to ca. 2 ml by rotary evaporation and elemental sulfur removed overnight with activated copper. The clean extract was separated into two fractions of different polarity (neutral and acidic) using Bond-elute (bond phase NH₂, 500 mg, 40 μm particle size) chromatography columns. A neutral lipid fraction was obtained by eluting with 15 ml DCM/2-propanol (2:1, v/v) and an acidic fraction with 15 ml of acetic acid (2%) in diethyl ether. Further separation of the neutral fraction into non-polar and polar sub-fractions was done with 0.5 g of alumina (activated, neutral, 0.05–0.15 mm particle size) in a pre-combusted Pasteur pipet. The non-polar fraction was obtained by eluting 4.5 ml of hexane/DCM (9:1, v/v) and the polar fraction with 3 ml of DCM/methanol (1:1, v/v). The acidic fraction was derivatized with BF₃ in methanol and the polar fraction with N,O-bis (trimethylsilyl) trifluoroacetamide (BSTFA).
The three lipid fractions (non-polar, acid, and polar fraction) were analyzed with gas chromatography mass spectrometry using a 6850 GC system coupled to a 5975 VL MSD with a triple axis detector (Agilent Technologies) operating in conditions previously described elsewhere (Sánchez-García et al., 2018 (link)). For the non-polar fraction, the oven temperature was programmed from 50 to 130°C at 20°C min^-1 and then to 300°C at 6°C min^-1 (held 20 min); for the acidic fraction, from 70 to 130°C at 20°C min^-1 and then to 300°C at 10°C min^-1 (held 10 min); and, for the polar fraction, the oven temperature program was the same as that for the acidic fraction, except that the oven was held for 15 min at 300°C. The injector temperature was 290°C, the transfer line was at 300°C, and the MS source at 240°C. Compounds identification was based on the comparison of mass spectra with reference materials, and their quantification on the use of external calibration curves of n-alkanes (C₁₀ to C₄₀), fatty acids methyl esters (FAME; C₈ to C₂₄), n-alkanols (C₁₀, C₁₄, C₁₈, and C₂₀), and branched isoprenoids (2,6,10-trimethyl-docosane, crocetane, pristane, phytane, squalane, and squalene). All chemicals and standards were supplied by Sigma Aldrich. The recovery of the internal standards averaged 69 ± 18%.

Leaf carotenoids, chlorophylls, and tocopherols were extracted in 2 mL Eppendorf tubes from 4 mg of freeze-dried leaf tissue, using 375 µL of methanol as the extraction solvent and a 25 µL of 10% (w/v) solution of canthaxanthin in chloroform (Sigma) as the internal standard. Tissue was lysed by adding 4 mm glass beads and grinding for 1 min at 30 Hz in a TissueLyser II (QIAGEN, Venlo, Netherlands) and the extraction was carried out by adding 400 µL of Tris-NaCl pH 7.5 and 800 µL of chloroform. Thoroughly mixed samples were centrifuged for 5 min at 13,000 rpm at 4 °C and the organic phase was transferred into a new tube and evaporated using a SpeedVac system (Eppendorf Concentrator plus, Hamburg, Germany). The extracted metabolites were then completely redissolved in 200 µL of acetone, filtered with 0.2 µm filters into amber-colored 2 mL glass vials and a 10-µL aliquot of each sample was then injected onto an Agilent Technologies 1200 series HPLC system (Agilent Technologies, Santa Clara, CA, USA). A C30 reverse-phase column (YMC Carotenoid, 250 × 4.6 mm × 3 µm, YMC CO., Kyoto, Japan) was used, with three mobile phases consisting of methanol (solvent A), water/methanol (20/80 v/v) containing 0.2% ammonium acetate (w/v) (solvent B), and tert-methyl butyl ether (solvent C). Metabolites were separated following the following gradient: 95% A, 5% B isocratically for 12 min, a step-up to 80% A, 5% B, 15% C at 12 min, followed by a linear gradient up to 30% A, 5% B, and 65% C by 30 min. The flow rate was maintained at 1 mL/min. The HPLC equipment was coupled to a Photometric Diode Array (PDA) detector (Santa Clara, CA, USA) allowing the detection of the full UV-visible absorption spectra of the different metabolites. Peak areas of chlorophylls at 650 nm and carotenoids at 472 nm were determined using Agilent ChemStation software. A fluorescence detector at 330 nm was used for tocopherol identification. The quantification of the compounds of interest was done by using a concentration curve built with a commercial standard (Sigma-Aldrich, Steinheim, Germany) [51 (link)]. For the rest of isoprenoid metabolites, approximately 7 mg of freeze-dried tissue were mixed with 500 µL of THF:MeOH (Analytical grade, Normapur) 1:1 buffered with 10% of water (v/v), thoroughly mixed, centrifuged, transferred to an amber vial, and injected into a Waters Acquity UPLC™ (Milford, MA, USA) coupled to a Waters Synapt G2 MS QTOF equipped with an atmospheric pressure chemical ionization (APCI) source. Prenyllipids were separated on an Acquity BEH C18 column (50 × 2.1 mm, 1.7 µm) under the following conditions: Solvent A = water; Solvent B = MeOH; 80–100% B in 4.0 min, 100% B for 2.5 min, re-equilibration at 80% B for 2.0 min. The flow rate was 500 µL/min, and the injection volume was 2.5 µL. Standards of HPLC grade (≥99.5%) were purchased from Sigma-Aldrich. PQ-9 and PC-8 standards were purified in house [52 ].

The isoprenoid quinones and cellular fatty acid composition were investigated by Techno Suruga Laboratory Co., Ltd. (Shizuoka, Japan), as described in a previous study [24 (link)]. Isoprenoid quinones were extracted as previously described by Tamaoka et al. [25 (link)]. The analysis of cellular fatty acids was carried out using cells grown in liquid DHB-CO₃-AF medium at 28 °C for 14 days, and the fatty acid profile was obtained according to the protocol of the Sherlock Microbial Identification System v6.0 (MIS, Newark, DE, USA) by accessing the TSBA6 database.