Lutein

Quercetin (Q4951 Sigma-Aldrich), Lutein (PHR1699 Sigma-Aldrich), Lycopene (PHR1770 Sigma-Aldrich), Epigallocatechin gallate (ECGC) (E009 TransMit) where dissolved in a solution of Dimethylsulfoxid (DMSO, 276855 Sigma-Aldrich) containing 1% of Tween 80 (P1754 Sigma-Aldrich) and mixed with bacteria to the following concentrations: Quercetin 100 µm, Lutein µM100, Lycopene 4.6 µM, ECGC 0.64 µM. Control worms were fed bacteria containing the same amount of solvent (0.5% DMSO plus 0.005% Tween 80) used to prepare the above compound. Serotonin (H9523 Sigma-Aldrich) treatment: worms were incubated in 200 µl of 10 mM Serotonin diluted in S-Basal. Imatinib (STI-57, SML1027 Sigma-Aldrich), was dissolved in H₂O and mixed with the bacteria to a final concentration of 1 µM.

Carotenoids were extracted by liquid–liquid extraction from plasma samples collected at 0 h and 24 h [32 (link)]. Chromatographic analysis of carotenoids was performed by HPLC-UV-DAD, using an HP 1100 HPLC system (Hewlett-105 Packard, Waldbronn, DE) containing a quaternary pump coupled to a DAD G1315B. The separation was carried out with Milli-Q water, methanol (MeOH) and methyl-tert-butyl ether (MTBE) (Panreac Quimica S.A., Barcelona, Spain), according to a procedure previously validated in our group [32 (link)]. A Waters RP column YMC Carotenoid S-5 µm (250 mm × 4.6 mm) and a precolumn YMC Guard Cartridge Carotenoid S-5 µm (20 mm × 4.0 mαm) were used.
Zeaxanthin (Extrasynthese, Genay, France), lutein, cryptoxanthin, α-carotene, β-carotene 9- and 13-cis-β-carotene (Sigma-Aldrich, St. Louis, MO, USA), lycopene (Fluka, Bucks, Switzerland), and 5-cis-lycopene (CaroteNature GmbH, Münsingen, Switzerland) were used as standards. These were pooled and prepared in synthetic human plasma (Sigma-Aldrich, St. Louis, MO, USA).
The sensitivity of each analyte was 0.703 µmol/L (lutein), 0.352 µmol/L (Zeaxanthin), 0.362 µmol/L (cryptoxanthin), 0.480 µmol/L (trans-β-apo-8’-carotenal), 0.745 µmol/L (13-cis-β-carotene), 0.373 µmol/L (9-cis-β-carotene and trans-β-carotene), and 0.186 µmol/L (trans and cis-lycopenes) [32 (link)].

HPLC grade acetonitrile, formic acid, ethyl acetate, petroleum ether, and diethyl ether were purchased from Sigma-Aldrich (Steinheim, Germany). Chlorophyll a and b standards were purchased from Sigma-Aldrich (Steinheim, Germany). Lutein, β-carotene, violaxanthin, and neoxanthin standards were purchased from Merck, Germany. Trolox, DPPH, and trypsin-EDTA were purchased from Sigma-Aldrich (Steinheim, Germany). Mueller–Hinton (MH) agar was purchased from Merck (Germany). DMSO (dimethyl sulfoxide) was purchased from Fluka (Buchs, Switzerland).

Tepals, cornas from S1 to S4 stages of Narcissus tazetta and transformed callus tissue, as well transformed Arabidopsis plants were collected for analysis the contents of flavonoids and (pro)anthocyanin. HPLC were performed to analysis the extracts of flavonoids metabolism according to the description by our previous publication (Ren et al. 2017 (link); Yang et al. 2021 (link)). The flavonoids and anthocyanin standards including naringenin, dihydroquercetin, kaempferol, quercetin, lutein, LeucoCyanidin, Catechin, and Cyanidin were purchased from Sigma-Aldrich (St. Louis, MO, USA), rutin and zeaxanthin were purchased from TCI (TCI Co., Ltd, Japan) and Sangon (Shanghai, China), respectively. All flavonoids and (pro)anthocyanin standards were prepared in MeOH/DMSO (1:1, v/v) and MTBE (containing 0.01% BHT), respectively, and stored at -20 °C before use.

Carotenoids were extracted and analyzed in the pulps of mature fruits of the four varieties (December and January for Navel oranges, and March and April for Valencia oranges) as described by Rodrigo et al. [26 (link)], using a Waters a liquid chromatography system (HPLC) equipped with a 600E pump, a photodiode array detector (DAD), model 2998 and Empower3 software (Waters, Barcelona, Spain). A C30 carotenoid column (250 × 4.6 mm, 5 μm) was coupled to a C30 guard column (20 × 4.0 mm, 5 μm) (YMC, Teknokroma, Spain). The carotenoids were identified by absorbance spectra and retention time; peaks were integrated at their individual maximal wavelength and their contents were calculated using the appropriate calibration curves of lycopene (Extrasynthese) for lycopene, neurosporene and δ-carotene, lutein (Sigma), β-carotene (Sigma), β-cryptoxanthin (Extrasynthese), zeaxanthin (Extrasynthese), anteraxanthin (CaroteNature) for anteraxanthin and mutatoxanthin and violaxanthin (CaroteNature) for violaxanthin isomers and luteoxanthin. Phytoene, phytofluene and ζ-carotene were previously purified by thin-layer chromatography from carotenoid extracts of Pinalate orange fruits [26 (link)]. The spectroscopic characteristics of all carotenoids detected in the pulps of Navel, Kirkwood, Valencia and Ruby oranges are shown in Table S1.