8453 uv vis spectrophotometer

The proline content was estimated according to the method of Bates et al. (1973) (link) (Quagliata et al., 2023 (link)). Briefly, 0.1 g fresh weight (FW) of tomato leaves were homogenized with 2 mL of 3% (w:v) 5-sulfosalicylic acid dihydrate. After a centrifugation step at 5000 rpm for 10 min, an aliquot (0.5 mL) of the supernatant was added to reaction tubes containing an equal volume of glacial acetic acid and acid-ninhydrin reagent (previously prepared by dissolving 1.25 g ninhydrin in 30 mL glacial acetic acid and 20 mL 6 M phosphoric acid). The reaction was conducted at 100°C for 1 h and stopped by cooling the samples in ice. The reaction mixture was extracted with 1.5 mL toluene and shaken vigorously for 20 sec. Subsequently, the chromophore containing toluene was separated from the aqueous phase and the absorbance read at 520 nm with an Agilent UV-Vis 8453 spectrophotometer (Santa Clara, CA, USA), using toluene as a blank. Calibration was done with 2 – 600 µL of a 1 mM L-proline (98.5 - 101.0%, pharma grade, PanReac AppliChem ITW Reagents S.R.L., Monza, Italy) stock solution, and the results were expressed as µmol g^-1 FW. Measurements were taken from 31 different plants.

A UV–Vis 8453 spectrophotometer (Agilent, Santa Clara, CA, USA) connected to a Brookfield TC-101 thermostat was used to determine the volume phase transition temperature (VPTT) of the particles obtained. Samples containing 3 mL of purified polymers were prepared. Some samples were diluted tenfold with deionised water due to their intense opalescence. The cuvette containing the polymer dispersion was placed in a spectrophotometer and thermostatted. The absorbance was recorded in the wavelength range 190–1100 nm and the values obtained at 480 nm were used. The first measurement was taken at 25 °C after the temperature had stabilised. After measuring all samples at this temperature, the temperature was increased by 1 °C until 45 °C was reached.

The content of pigments (chlorophyll a, chlorophyll b, and carotenoids) was measured in leaves of tomato plants sampled 48 h after the chilling exposure, following the method of Prodhan et al. (2017) (link) with slight modifications. Four mL of chilled methanol were added to 0.050 g FW of leaf material. The mixture was homogenized and incubated for 30 min in the dark at 4°C. Afterwards, the samples were centrifuged (PK110 centrifuge, Alc International S.r.l., Cologno Monzese, MI, Italy) at 3500 rpm for 20 min. The absorbance of supernantants were measured at 470, 653 and 666 nm with an Agilent UV-Vis 8453 spectrophotometer (Santa Clara, CA, USA). The specific absorption coefficient in methanol was used to calculate chlorophyll a and b and total carotenoid contents in leaves. The results were expressed as mg g^-1 FW (Lichtenthaler and Wellburn, 1983 (link)).

The proline content was estimated according to the method of Bates et al. (1973) (link) (Quagliata et al., 2023 (link)). Briefly, 0.1 g fresh weight (FW) of tomato leaves were homogenized with 2 mL of 3% (w:v) 5-sulfosalicylic acid dihydrate. After a centrifugation step at 5000 rpm for 10 min, an aliquot (0.5 mL) of the supernatant was added to reaction tubes containing an equal volume of glacial acetic acid and acid-ninhydrin reagent (previously prepared by dissolving 1.25 g ninhydrin in 30 mL glacial acetic acid and 20 mL 6 M phosphoric acid). The reaction was conducted at 100°C for 1 h and stopped by cooling the samples in ice. The reaction mixture was extracted with 1.5 mL toluene and shaken vigorously for 20 sec. Subsequently, the chromophore containing toluene was separated from the aqueous phase and the absorbance read at 520 nm with an Agilent UV-Vis 8453 spectrophotometer (Santa Clara, CA, USA), using toluene as a blank. Calibration was done with 2 – 600 µL of a 1 mM L-proline (98.5 - 101.0%, pharma grade, PanReac AppliChem ITW Reagents S.R.L., Monza, Italy) stock solution, and the results were expressed as µmol g^-1 FW. Measurements were taken from 31 different plants.

The content of pigments (chlorophyll a, chlorophyll b, and carotenoids) was measured in leaves of tomato plants sampled 48 h after the chilling exposure, following the method of Prodhan et al. (2017) (link) with slight modifications. Four mL of chilled methanol were added to 0.050 g FW of leaf material. The mixture was homogenized and incubated for 30 min in the dark at 4°C. Afterwards, the samples were centrifuged (PK110 centrifuge, Alc International S.r.l., Cologno Monzese, MI, Italy) at 3500 rpm for 20 min. The absorbance of supernantants were measured at 470, 653 and 666 nm with an Agilent UV-Vis 8453 spectrophotometer (Santa Clara, CA, USA). The specific absorption coefficient in methanol was used to calculate chlorophyll a and b and total carotenoid contents in leaves. The results were expressed as mg g^-1 FW (Lichtenthaler and Wellburn, 1983 (link)).

High performance liquid chromatography (HPLC) analysis was performed on a Hewlett Packard/Agilent series 1100 (Agilent, Santa Clara, CA, USA) equipped with an analytical C18 reverse phase column (Kinetex, 75 × 4.6 mm, 2.6 μm, Phenomenex, Torrance, CA, USA). Nuclear magnetic resonance (NMR) studies were performed on a Varian 500 system (400 MHz). The sizes and polydispersity of micelles were determined on a Delsa Nano C particle analyzer (Beckman Coulter, CA, USA). Fluorescence microscopy was conducted on a FSX 100 (Olympus, PA, USA). 8453 UV–Vis spectrophotometer (Agilent, CA, USA) was used to acquire UV–Vis spectra and Cary Eclipse fluorescence spectrophotometer was used to measure fluorescence. For transmission electron microscopy (TEM) image, a 2.0 μL aliquot of the micelle solution was deposited on a copper grid coated with a carbon film. The sample was dried at room temperature and then imaged on a Tecnai G2 Spirit BioTWIN transmission electron microscope, operating at 100 kV (note that this drying procedure employed in TEM analysis may cause considerable changes in the size and shape of the micelles since they only exist in solution).