Uv 1800pc spectrophotometer

The α-amylase assay was conducted as described by Oseguera-Toledo et al. (21 (link)) with minor modifications. The assay mixture containing 500 μL of the protein hydrolysates at different concentrations (0.1, 0.5, 1, 1.5, 2 and 2.5 mg/mL) and 500 μL of α-amylase from B. subtilis (1 U/mL) was pre-incubated in test tubes at 37 °C for 10 min in a water bath (model HH-4; Wincom Company Ltd). Then, 500 μL of 1% starch prepared in 0.02 mM sodium phosphate buffer at pH=6.9 with 6.7 mM NaCl were added, and the mixture was incubated for another 15 min at 37 °C. The reaction was terminated by adding 500 μL of 3,5‐dinitrosalicylic acid (DNS) colour reagent to each test tube and placed in boiling water bath for 10 min. The reaction mixture was cooled and diluted with 5 mL of ultrapure water. The absorbance was measured at 540 nm using a UV-1800PC spectrophotometer (Shanghai Mapada Instruments Co., Ltd). Control was sodium phosphate buffer (pH=6.9) and blank contained the sample and buffer without the enzyme. Acarbose (1 mg/mL) was used as a positive control. The inhibition (%) was calculated with the following formula:

Hydroxyl radical scavenging activity of the hydrolysates was analysed according to a modified method of Chi et al. (12 (link)). In this study, sample concentration was 0.5, 1, 1.5 and 2 mg/mL. The mixtures were kept in water bath at 25 °C for 90 min and the absorbance was measured at 536 nm by a UV-1800PC spectrophotometer (Shanghai Mapada Instruments Co., Ltd.). The reaction mixture without antioxidant was used as the negative control, and a mixture without H₂O₂ was used as the blank. The hydroxyl radical scavenging activity (HRSA) was calculated by the following formula:
where A_s, A_n and A_b are the absorbances of the sample, negative control and the blank after the reaction, respectively. BHT was used as positive control.

The adsorption capacities of RWT-PC, NaOH-PC, CaO-PC, and PC after Na₂CO₃ activation (Na₂CO₃-PC) for MG were measured via adsorption experiments. Forty milligrams of X-PC (X = RWT, NaOH, CaO, Na₂CO₃) were added to 40 mL of MG solution with an initial concentration of 100–1000 mg/L. The mixture was then oscillated at 130 rpm for 48 h at 301 K for adsorption. After adsorption, the MG solution was filtered and its concentration measured at 618 nm using a UV-1800PC spectrophotometer (Mapada, Shanghai, China). The equilibrium adsorption capacities (q_e) of RWT-PC, NaOH-PC, CaO-PC, and Na₂CO₃-PC for MG dyes were calculated using Equation (1).
$$q_e=\frac{\left(C_O-C_e\right)\times \mathrm{V}}{\mathrm{W}}$$

Chlorophylls and carotenoids determination was carried out according to the method described by Luo et al. (2019) (link) with minor modifications. Chlorophylls and carotenoids were extracted from 50 mg of leaves in 5 mL of dimethyl sulfoxide, after incubation at 65°C for 20 min (until the leaves turned white). The extraction solution was measured at 470 nm, 649 nm, and 665 nm using the UV-1800PC spectrophotometer (MAPADA, Shanghai, China). Content of chlorophylls (C_T) and carotenoids (C_c) were calculated using the following equations (“V” represents the final volume of the reaction and “m” represents the mass of leaves used for metabolites extraction):
Anthocyanins were extracted and determined using the method of Wang et al. (2019a) (link) with little modification. Briefly, 50 mg of frozen leaves was grounded into powder in liquid nitrogen, sonicated with 3 ml of 0.1% methanol hydrochloride for 1 h, and then shaken overnight. After centrifugation at 2,500 g for 10 min, 1 ml of the supernatant was mixed with 1 ml of water and the mixture was further mixed with 1ml of chloroform to remove chlorophyll. The resulted solution was measured at 530 nm for anthocyanin determination.

At the three-leaf stage, the leaf samples (0.1 g) of WT and oswhy1-1 mutant were collected and cut into 1 cm segments and soaked in 10 ml 80% ethanol for 48 h at room temperature in the dark. The supernatant was measured at 662 nm (maximum absorption peak of Chl a), 646 nm (maximum absorption peak of Chl b), and 470 nm (maximum absorption peak of Car) light using a UV-1800PC spectrophotometer (Mapada, China). The concentrations of Chl a, Chl b, and Car were calculated using the methods of Arnon (1949 (link)) and Wellburn (1994 (link)) (Arnon 1949 (link); Wellburn and Alan 1994 (link)). Three biological replicates were carried out.

The Chlorophyll a (Chla), chlorophyll b (Chlb), carotenoid (Car) and total chlorophyll (Chl) content was measured. In brief, we obtained WT and oscaf2 mutant leaves (0.2 g) at the seedling stage. The leaves were cut and soaked in 5 ml 95% ethanol for 48 h under dark conditions. Spectrophotometric measurements were conducted using a UV-1800PC spectrophotometer (Mapada, China) at 470 nm, 649 nm, and 665 nm. According to previous methods [36 ], we calculated the Chla, Chlb, Car and Chl content in WT and oscaf2 mutant leaves.

Chl fluorescence was measured with a Dual-PAM-100 Chl fluorescence photosynthesis analyzer (Walz, Germany) using 3 ml culture, grown under NL or -C conditions for 8 h at room temperature in darkness, according to the manufacturer's instructions, at which point the cells were in the exponential growth phase. All cultures were enriched to OD₇₃₀ 1.0 by centrifugation at 2,500 g (25°C, 5 min). Absorbance of whole cells was measured with a UV-1800 PC spectrophotometer (MAPADA, China). All values represent the means of five biological replicates.