Fluorescence detector system

One hundred microliters of seed culture incubated overnight at 37 °C in LB medium containing 50 mg/L kanamycin was inoculated into 1 mL LB medium containing 50 mg/L kanamycin and incubated at 37 °C until the optical density of the E. coli culture at 600 nm (OD600) reached 1.0. After the addition of 1 mM isopropyl-β-D-thiogalactopyranoside and 1 mM 5-methoxytryptamine, the culture was grown and shaken at 250 rpm at varying temperatures such as 28, 37, and 42 °C for the indicated time periods. The resulting cultures were centrifuged at 11,500× g for 5 min and separated into cell pellet and medium (supernatant) fractions. The medium fractions (0.2 mL) were mixed with 0.2 mL of 100% methanol. The resulting 10 µL aliquots were subjected to high-performance liquid chromatography (HPLC) using a fluorescence detector system (Waters, Milford, MA, USA) as described previously [34 (link)].

Frozen rice samples (0.1 g) were ground into powder in liquid nitrogen using the TissueLyser II (Qiagen, Tokyo, Japan) and extracted with 1 mL of chloroform for 24 h at 4 °C. The chloroform extracts (200 µL) were completely evaporated and dissolved in 0.1 mL of 40% methanol, and 20 µL aliquots were subjected to high-performance liquid chromatography using a fluorescence detector system (Waters, Milford, MA, USA). Melatonin was detected by emissions at 348 nm, using 280 nm excitation. All measurements were taken in triplicate.

Melatonin concentration was measured according to Byeon et al. [59 (link)] with a few modifications. Leaf samples (0.2 g) were pulverized to powder in liquid nitrogen and extracted with 10 mL chloroform for 1 h and ultrasound for 15 min. Then the extracts were centrifuged at 10,000× g for 15 min at 4 °C and evaporated with nitrogen gas to dry. The substance was dissolved in 1 mL 80% methanol and filtered by a membrane filter (0.22 µm). 10 µL samples were analyzed by HPLC system with a fluorescence detector system (Waters, Milford, MA, USA). All measurements were determined in triplicate.

Seven-day-old seedlings were transferred to 50 mL polypropylene conical tubes containing 25 mL water with 0.5 mM cadmium (CdCl₂) or varying concentrations of aluminum (AlCl₃). These seedlings were incubated for three days under continuous light as described previously [32 (link)]. The rice samples (shoot parts) were frozen in liquid nitrogen and pulverized to a powder using a TissueLyser II instrument (Qiagen, Tokyo, Japan). The powder (100 mg) was extracted with 1.5 mL chloroform for 1 h at room temperature. The chloroform extracts were evaporated until dry and dissolved in 0.1 mL 42% methanol. Aliquots of 10 µL were subjected to HPLC with a fluorescence detector system (Waters, Milford, MA, USA), as described previously [32 (link)].

Rice samples (100 mg) were frozen in liquid nitrogen and pulverized to a powder using TissueLyser II (Qiagen, Tokyo, Japan), and extracted with 1 mL of chloroform at room temperature as described previously [70 (link)]. In brief, the chloroform extracts (200 µL) were completely evaporated and dissolved in 0.1 mL of 40% methanol, and 10-µL aliquots were subjected to high performance liquid chromatography coupled with a fluorescence detector system (Waters, Milford, MA, USA). Melatonin was detected at 280 nm (excitation) and 348 nm (emission). The detection limit and recovery rate of melatonin were approximately 0.25 ng/g FW and 90%, respectively, using this analytical method. All measurements were made in triplicate.

Homozygous T₂ transgenic rice seeds were used in further studies. Dehusked wild-type and transgenic rice seeds were sterilized with 2% NaOCl for 50 min, thoroughly rinsed with sterile distilled water, and sown on half-strength Murashige and Skoog (MS) medium under cool daylight fluorescent lamps (60 μmol m⁻² s⁻¹) (Philips, Amsterdam, The Netherlands) under a 14 h light/10 h dark photoperiod at 28 °C/24 °C (day/night). Seven-day-old seedlings were used in further experiments. For mannitol (Sigma-Aldrich, St. Louis, MO, USA) treatment, surface-sterilized rice seeds were sown and grown on half-strength MS medium containing various concentrations of mannitol. Melatonin contents were measured in frozen samples (0.1 g) that were pulverized to a powder in liquid nitrogen using the TissueLyser II (Qiagen, Tokyo, Japan). The sample powders were extracted with 1 mL chloroform, followed by centrifugation for 10 min at 12,000× g, and the supernatants (200 µL) were evaporated and dissolved in 0.1 mL 40% methanol. The resulting 10 µL aliquots were subjected to high-performance liquid chromatography (HPLC) with a fluorescence detector system (Waters, Milford, MA, USA) as described previously [19 (link)]. Melatonin was eluted after about 31 min under the HPLC conditions. The measurements were performed in triplicate.

Frozen samples (0.1 g) were ground in liquid nitrogen with the use of the TissueLyser II (Qiagen, Tokyo, Japan) and extracted with 1 mL of chloroform. The chloroform extracts were centrifuged for 10 min at 12,000× g, and the supernatants (200 μL) were completely evaporated and dissolved in 0.1 mL of 40% methanol, and 20-μL aliquots were subjected to HPLC using a fluorescence detector system (Waters, Milford, MA, USA) as described previously [27 (link)]. In brief, melatonin was detected at 280 nm (excitation) and 348 nm (emission) on a Sunfire C18 column (Waters 4.6 × 150 mm) in the following gradient elution condition: from 42% to 50% methanol in 0.1% formic acid for 27 min, followed by isocratic elution with 50% methanol in 0.1% formic acid for 18 min at a flow rate of 0.15 mL/min. All measurements were performed in triplicate.