Naringenin chalcone

Wild-type rice (Oryza sativa L. subsp. Japonica cv. Dongjin) seeds were sterilized with 50% bleach for 30 min and germinated on Murachige and Skoog medium (Duchefa Biochemie, Haarlem, The Netherlands) in a growth chamber with a 16 h light/8 h dark photoperiod at 28 °C for two weeks. The rice seedlings were transferred to soil and grown in a greenhouse at 28 °C during the day. Shoot and root samples were collected from two-week-old rice seedlings. Leaves, stems, and roots were obtained from eight-week-old rice plants during the vegetative growth period. Panicles were harvested from 14-week-old rice plants. Eight-week-old rice plants were irradiated with UV light to examine the UV stress response. UV treatment and HPLC analysis were carried out according to the methods described by Park et al. [25 (link)].
Naringenin chalcone and isoliquiritigenin were purchased from Sigma-Aldrich (St. Louis, MO, USA). E. coli BL21(DE3) and Rosetta 2(DE3) strains were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Ni-NTA Agarose beads and CM Sepharose Fast Flow resin were purchased from Qiagen (Hilden, Germany) and Cytiva (Marlborough, MA, USA), respectively. Restriction enzymes were bought from New England Biolabs (Ipswich, MA, USA) and Enzynomics (Daejeon, Korea). Reagents for buffers and media were obtained from Sigma-Aldrich and Duchefa Biochemie.

The major phenylpropanoids were extracted from the exocarp of tomato and the 0.2 g skin samples were ground to powder in liquid nitrogen and extracted overnight at −20 °C in 2 mL methanol with 100% chromatographic grade purity (Sigma-Aldrich, Saint Louis, MO, USA). The first 2 h were shaken and mixed every 15 min for full extraction. The sample was centrifuged at 4 °C and 4000 g for 20 min. The supernatant was filtered with 0.22 µm microporous membrane and stored in dark at −20 °C. The phenylpropanoids were quantified by HPLC (high-performance liquid chromatography, Agilent Technologies 1200 series, Beijing, China) with a chromatographic column (Agilent Technologies ZORBAX SB-C18 4.6 × 250 mm). Aqueous phase A was 0.1% acetic acid solution; organic phase B was pure methanol. The gradient as follow: t = 0.0 min, 20% B; t = 10 min, 30% B; t = 25 min, 90% B; t = 27 min, 90% B; t =28 min, 20% B; t = 32 min, 20% B, each sample was injected 10 µL and the flow rate was 1 mL/min. Detection by ultraviolet (UV) chromatograms was recorded at 325 nm and the column temperature was 35 °C [30 (link)]. All phenylpropanoid standards, rutin, naringenin chalcone and kaempferol rutinoside were obtained from Sigma-Aldrich.

The major flavonols in tomato fruits of Micro-Tom, CSl09-03 and Sheng Nv-Guo were extracted from freeze-dried tomato test specimens using 70% methanol from Sigma (dry-weight basis), and the major flavonols from the ten tomato cultivars numbered 1 to ten were extracted from skin samples (0.2 mg) using 2 ml of 100% methanol from Sigma (http://www.sigmaaldrich.com/) (fresh-weight basis). The flavonols were quantified by HPLC (high-performance liquid chromatography, Agilent Technologies 1200 series) with acolumn (Agilent Technologies ZORBAX SB-C18 4.6*250 mm). A gradient elution was performed with solvent A consisting of 3% acetonitrile and 10% formic acid and solvent B consisting of acetonitrile (50%) and formic acid (10%), with the following elution program: 0 min 4% B, 20 min 20% B, 35 min 40% B, 40 min 60% B, 45 min 90% B, 55 min 4% B, flow rate of 1 mL/min^{7 (link)}. Detection by ultraviolet (UV) chromatograms was recorded at 325 nm. All flavonol standards, rutin, kaempferolrutinoside and naringenin chalcone were obtained from either Sigma-Aldrich or Extrasynthèse (Genay, France).