Uv 3310 spectrophotometer

Soluble sugars were determined according to the method described by Du et al. (2020) (link). The fresh leaves (0.5 g) were extracted in the boiling water bath for 30 min. Its remaining residue was extracted twice more, and the extraction solutions were combined in a volumetric flask (100.0 mL). Then, the test tube with extraction solution (0.5 mL) added cocktail consisting of 0.5 mL of anthrone-ethyl acetate reagent, 5.0 mL of concentrated sulfuric acid, and distilled water (1.5 mL). After shaking, the test tube was immediately put into the boiling water bath for 10 min. After cooling, absorbance was determined three times by the UV-3310 spectrophotometer (Hitachi, Japan) at 620 nm wavelength. The soluble sugar content was calculated by a standard curve (glucose) and expressed as mg·g⁻¹ FW.

YL001 and ΔcpxR cells were grown overnight in TSB medium at 28°C by shaking at 180 rpm. Then, the cells were resuspended in TSB medium with an initial OD₆₀₀ value of 0.01, respectively. Under the identical conditions, the growth rates were monitored every 6 hr during the 72‐hr period using a UV‐3310 spectrophotometer (Hitachi, Japan). Before testing, each sample solution was fully dispersed using a moderate ultrasonic amplitude (50%) for 5 min to avoid the probable cell aggregation. Each experiment was performed in triplicate.

Malic enzyme activity in P. tricornutum was measured using an NADPH-ME kit (Solarbio, Beijing, China) according to manufacturer instructions. The optimum reaction system was prepared as followed: 50 mM, pH 7.5 Tris–HCl, 1 mM MgCl₂, 0.5 mM NADP⁺, 10 mM L-malate. Soluble protein concentration was quantified using a Bradford assay kit (Genmed Scientifics, Shanghai, China). ME activity was determined by monitoring the change in absorbance at 1-min intervals continuously at 340 nm using a UV-3310 spectrophotometer (Hitachi). One unit of ME activity was defined as 1 μM NADPH generated by 1 mg protein per minute in the reaction system:
where A1 is the initial absorbance, A2 is the absorbance after the reaction, 6.22 represents the extinction coefficient per mM NADPH, t is the reaction time (1 min), l is the path length of the cuvette (1 cm), V1 is the total reaction volume (900 μL), V2 is the volume of ME solution (30 μL), and C is the concentration of protein (mg/mL).

Soluble protein was measured using the UV-3310 spectrophotometer (Hitachi, Japan) at 595 nm. Briefly, leaves (0.5 g) were ground to homogenate with 5.0 mL of distilled water, and then the homogenate was centrifuged at 12, 000 × g for 20 min at 4 °C. Supernatant (1.0 mL) was collected and added to 5.0 mL of Coomassie Brilliant Blue G-250 solution in the test tube at 30 °C for 30 min for the soluble protein determination. The absorbance of the reaction solution was measured with a spectrophotometer at 595 nm. Bovine serum albumin was used as a standard to quantitatively analyze the content of soluble protein, which was expressed as mg·g⁻¹ FW (Zou et al., 2017 (link)).

Chlorophyll content was determined according to the method of Min et al. (2019) (link), with some modifications. Ten fresh leaves were collected from each plot and washed as test samples. The samples (0.3 g) were then ground to homogenate in a mortar with quartz sand, calcium carbonate powder, and 5.0 ml of pure acetone. The extracting reaction was performed by adding 5.0 mL of 80% (w/v) acetone to the homogenate for 10 min under dark conditions. The extraction solution was centrifuged using the Centrifuge 5424 (Eppendorf AG, Germany) at 20, 000 g for 30 min at 4 °C. Supernatant was collected, and its absorbance was recorded by the UV­3310 spectrophotometer (Hitachi, Japan) at 663 nm and 645 nm, respectively. There were three replicates for each treatment. The chlorophyll content (chlorophyll a and chlorophyll b) was calculated using the equation of Min et al. (2019) (link). The results are expressed on a dry weight basis (mg·g⁻¹ FW).

Antioxidant enzyme activities, including superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), were determined following the protocol of Shehzad et al. (2020) (link) and expressed as U·g⁻¹ FW. Firstly, leaves (0.5 g) were fully homogenized with 2.0 mL of pre-cooled phosphate buffer (50 mM, pH 7.8) and 7.0 mL of 1.0% (w/w) polyvinylpyrrolidone. The homogenate was then centrifuged at 8, 000 × g for 15 min at 4 °C to collect supernatant for enzyme analysis. The absorbance was monitored every 20 s by a UV-3310 spectrophotometer (Hitachi, Japan) at 240 nm wavelength. The CAT activity was evaluated by measuring the decomposition of H₂O₂. The POD activity was assessed by monitoring the absorbance at 470 nm using guaiacol. To assay the SOD activity, the absorbance was measured at 570 nm after 20 min of chromogenic reaction. All enzymes’ activities were expressed as units U·g⁻¹·FW.