C8 column

Peptides were separated by reversed-phase chromatography on a HPLC (Waters, USA) system equipped with a C8 column (250 mm × 4.6 mm × 5 mm; Waters) and photodiode array detector (Spectra System Thermo Scientific, USA) A gradient elution was applied from 100 to 0% A in 56 min at flow rate of 1 mL/min. A binary mobile phase consisted of solvent A: 0.065% trifluoroacetic acid [TFA] in water/acetonitrile [ACN] 98:2, and solvent B: 0.065% TFA in water/ACN 35:65. Detection was performed at 280 nm and temperature was set at 40 °C. The injection volume was 200 µL. Fractions were manually collected every 1 min in Eppendorf tubes and protein content was evaluated by the Bradford assay. Finally, samples were lyophilized (Labconco DrySystem/freezone 4.5) and bioactivity analysis was carried out only on the lyophilized samples containing protein.

For the pigment analysis, the leaves were collected from the sprl1 mutant and its wild type at the seedling stage and the heading stage, respectively. The pigments were extracted from 0.2 g of fresh leaves with 80% acetone in the dark at 4 °C for 48 h. The contents of chlorophyll (Chl) and carotenoids (Caro) were measured at wavelengths of 470 nm, 646 nm, and 663 nm using the BIOMATE 3S UV-Visible Spectrophotometer (Thermo scientific, Waltham, MA, USA) and were calculated according to the method of Lichtenthaler and Wellburn [52 (link)]:


For the Proto IX analysis, 0.1 g fresh leaves at the seedling stage were homogenized in 1 mL methanol/acetone/0.1 M NaOH (9:10:1, v/v/v), and the homogenates were centrifuged at 7197 g (Eppendorf 5430R; 7830 rpm) at 1 °C for 20 min. To oxidize the Protogen IX into Proto IX, 5 μL of 1 M acetic acid and 5μL of 2-butanone peroxide was added to 200 μL supernatant [28 (link)]. Then, the Proto IX was analyzed by HPLC on a C8 column (4.6 × 150 mm, 3.5μm; Waters) according to the method of Wang [53 (link)]. The elution profiles were detected by fluorescence, with excitation at 405 nm and emission at 625 nm [28 (link)]. The Proto IX was quantified by using the Proto IX standard (Frontier Scientific, Logan, UT, United States).

Extracted lipids were resuspended in organic buffer (methanol containing 1 mM ammonium formate and 0.2% formic acid). A two-buffer mobile system, aqueous and organic was used. Aqueous buffer contained 2 mM ammonium formate and 0.2% formic acid. From each sample, 5 µl was injected by the Autosampler, and mobile buffer was pumped at a flow rate of 0.3 ml min⁻¹ to the HPLC fitted with the C8 column (Waters, MA, USA). SL species were detected by multiple reaction monitoring (MRM) methods using QTRAP® 4500 (SCIEX, USA) mass spectrometer. The MRM scans used were described earlier by Kumar et al. (2021 (link)).

RP-HPLC was used to analyze the protein in the top phase before and after purification. The conditions for RP-HPLC were as follows: Waters C₈ column (4.6 mm × 150 mm), 0.05% TFA in acetonitrile as mobile phase A, 0.05% aqueous TFA as mobile phase B, flow rate of 1.00 mL/min, and detection wavelength of 280 nm. Gradient elution was programmed as follows: the concentration of mobile phase A was increased from 7% to 70% in 17 min and decreased to 7% before 22 min [27 (link)].

The 20 μL plasma sample was extracted with 180 μL methanol containing internal standards in a 96-well plate with a filter membrane. After centrifugation, 50 μL of the filtrate was transferred to a microtiter plate and diluted 4 times for analysis [24 (link)]. The chromatographic separation was achieved on a C8 column (2.1 × 100 mm i.d., 1.7 μm, Waters Corp., Milford, MA, USA) at 45 °C with mobile phase A (0.1% formic acid in water, v/v) and mobile phase B (acetonitrile), and the flow rate was set at 0.5 mL/min. The gradient profile was as follows: linear gradient 0–2.5% B, 0.5 min, 2.5–12% B, 2 min, 12–36% B, 0.5 min, and keep 36% B, 1 min, 36–100% B, 0.2 min, and keep 100% B, 1.3 min. The column was then re-equilibrated for 1.4 min. The parameters of MS were as follows: capillary voltage 1 kV; desolvation gas flow 1000 L/h; desolvation temperature 550 °C; source temperature 150 °C. Acylcarnitines were quantified in positive electrospray ionization mode.