Empower software v 3

Plant pigments were extracted and subsequently analysed by Acquity UPLC™ H-Class System (Waters, Milford, MA, USA) equipped with an autosampler, quaternary solvent manager and a photodiode-array detection detector as described in Moreno et al. (2020) (link). Five biological replicates per line were measured and data were analysed with Empower software (v. 3, Waters, Manchester, UK).

LC-MS analysis was performed using an Alliance chromatographic system coupled to a ZQ4000 mass spectrometer and a SunFire C18 column (3.5 mm, 2.1 × 150 mm; Waters). Solvents were the same as those used in the UPLC analyses and elution was performed with an initial isocratic hold with 10% acetonitrile for 4 min followed by a linear gradient from 10 to 88% acetonitrile over 26 min, at 0.25 mL min⁻¹. After the LC phase, MS was carried out by electrospray ionization in the positive mode, with a capillary voltage of 3 kV and a cone voltage of 20 and 50 V. Detection and spectral characterization of the peaks was performed by photodiode array detection using Empower software v.3 (Waters) to extract bi-dimensional chromatograms at different wavelengths, normally within the range between 200 and 500 nm depending on the spectral characteristics of the desired compound.

Metabolites from the Streptomyces R5A cultures (inoculated with 10⁶ live spores) grown during 96 h (i.e., stationary phase) were extracted using the following procedure. Ethyl acetate was added to the samples (containing both supernatants and biomass) at a 1:1 ratio (20 mL:20 mL) and maintained in constant mixing for 2 h. The organic phase (≤20 mL) was separated from the aqueous phase by centrifugation and collected for further evaporation using a SpeedVac. The resulting extracts were re-suspended in 200 µL of methanol. The cell extracts were analyzed by reversed-phase chromatography using acetonitrile and water containing 0.1% trifluoroacetic acid (TFA) as solvents in an Acquity UPLC instrument fitted with a BEH C18 column (1.7 mm, 2.1 × 100 mm; Waters). Samples were eluted with 10% acetonitrile for 1 min, followed by a linear gradient from 10 to 100% acetonitrile over 7 min, at a flow rate of 0.5 mL min⁻¹ and a column temperature of 35 °C. Detection and spectral characterization of the peaks obtained from UPLC was performed by photodiode array detection using Empower software v.3 (Waters) to extract bi-dimensional chromatograms at different wavelengths.