Ultimate 3000 system

The crude enzyme (0.5 ml) was added into 50 ml Tris–HCl (50 mM, pH 7.0) containing 0.5% sodium alginate and incubated at 50°C for 24 hr. The reaction was stopped by heating in boiling water for 10 min. Then samples from this enzymatic hydrolysate and seaweed fermentation broth (SFE‐12) were treated by Sevag method to remove proteins (Staub, 1965) and filtered with 0.22 μm filter. Chromatographic analysis of alginate oligosaccharides was performed on UltiMate 3000 system equipped with a PDA detector (Waters 2996, Waters, USA). A Superdex 30 column (10 mm × 300 mm, GE Healthcare, USA) maintained at 25°C was used as the separation channel. Isocratic elution was performed at 25°C with mobile phase consisting of 0.1 mol/L NaCl at a constant flow rate of 0.2 ml/min. The injection volume for standards and samples were 20 μl. UV detection was carried out at 230 nm. The mannuronic acid sodium salt dimer, trimer, and tetramer (5 mg/ml) were used as standards.

Proteins were extracted from the same 12 pooled samples used for transcriptome sequencing. The plant material was pulverized in liquid nitrogen and further homogenized in pyrolysis solution (7M urea, 2% SDS, 0.1% PMSF, 65 mM DTT) with ultrasonication. Supernatants were centrifuged (14 000 rpm, 30 min, 4 °C) and the protein contents were measured by the BCA method (Walker, 1994 (link)). After that, the proteins were reduced and alkylated using 50 ul DTT (1 h, 55 °C) and 5 ul of 20 mM iodoacetamide (IAA; 1 h, room temperature, in the dark), respectively. The proteins were then precipitated for 2 h with 300 ul of precooled acetone and hydrolyzed with trypsin (Promega) overnight. The resulting peptides were dissolved in buffer A (buffer A: 20 mM ammonium formate aqueous solution, adjusted to pH 10.0 by ammonia water) and separated using a reverse column (XBridge C18 column, 4.6 mm x 250 mm, 5 um) connected to an Ultimate 3000 system (Waters Corporation, MA, USA) using 5% to 45% solution B (20 mM ammonium formate with 80% ACN, adjusted with ammonia to pH 10.0) for separation. Fractions were lyophilized before analysis.

Liquid chromatography was performed on a Thermo Scientific UltiMate 3000 system equipped with a 2.1 x 50 mm XBridge^™ OST C18 column packed with 2.5 μm particles (Waters Ltd., UK), using Chromeleon 7 software (Version 7.1.1.1127). The gradient system used for LC analysis consisted of 100 mM triethylammonium bicarbonate (TEAB) as Buffer A and 40% acetonitrile (ACN) in water (HPLC grade, Fisher Scientific, UK) as Buffer B. For Buffer A, a 1 M pre-formulated TEAB solution was purchased from Sigma-Aldrich (UK), and diluted to the required concentration with HPLC grade water. The gradient was ramped from 90% A at 0 min to 55% A at 18 min, then to 20% A at 22 min, and finally to 10% A at 23.5 min. UV absorbance was monitored at 254 nm.

A Thermo Ultimate 3000 system equipped with an ACQUITY UPLC^® HSS T3 (150 × 2.1 mm, 1.8 µm, Waters, Milford, DE, USA) column was employed to separate the metabolites. The temperatures of autosampler and column were maintained at 8 °C and 40 °C, respectively. The sample volume was 2 µL. Mobile phase in negative-ion mode was solvent A (5 mM ammonium formate in water) and solvent B (acetonitrile). Mobile phase in positive-ion mode was solvent C (0.1% formic acid in water) and solvent D (0.1% formic acid in acetonitrile). The elution procedure was as follows: 2% B/D (0~1 min), 2%~50% B/D (1~9 min), 50%~98% B/D (9~12 min), 98% B/D (12~13.5 min), 98%~2% B/D (13.5~14 min), 2% D in positive model (14~20 min) or 2% B in negative model (14~17 min). The flow rate was set as 0.25 mL/min. The separated metabolites were then analyzed on a Thermo Q Exactive mass spectrometer with electrospray ionization (ESI) system [24 (link)]. The spray voltages applied were 3.8 kV in positive mode and 2.5 kV in negative mode. Sheath gas and auxiliary gas were set as 30 and 10 arbitrary units, respectively. The capillary temperature was maintained at 325 °C. The m/z scan range was 81 to 1000 for full scan at a mass resolution of 70,000 [24 (link)]. Data-dependent acquisition (DDA) and dynamic exclusion were performed according to the procedures as described [24 (link),25 (link)].