4 pobn

Spin trapping assays with the spin probe 4-pyridyl-1-oxide-N-tert-butylnitrone (4-POBN; Sigma-Aldrich, St. Louis, USA) to detect the formation of hydroxyl radicals^{31 (link)} were carried out using cyanobacterial cells (OD₇₃₀ = 0.65) in 10 mM sodium phosphate buffer (pH 7.0) containing 50 mM 4-POBN, 4% (v/v) ethanol, 50 µM Fe-EDTA, and 50 µM MV. After a 30-min incubation in the light (10 µmol m⁻² s⁻¹), the samples were centrifuged at 7000g for 1 min, and the supernatants were frozen in liquid nitrogen and stored at − 80 °C for electron paramagnetic resonance (EPR) spectra analysis. The EPR spectra were recorded at room temperature in a standard quartz flat cell using an ESP-300 X-band spectrometer (Bruker, Rheinstetten, Germany). The following parameters were used: microwave frequency, 9.73 GHz; modulation frequency, 100 kHz; modulation amplitude, 1 G; microwave power, 6.3 milliwatt; receiver gain, 2 × 104; time constant, 40.96 ms; number of scans: 4.

Electron paramagnetic resonance (EPR) spin‐trapping assays for determining H₂O₂ were carried out using the spin trap 4‐pyridyl‐1‐oxide‐N‐tert‐butylnitrone (4‐POBN) (Sigma‐Aldrich). Leaves were vacuum‐infiltrated with a buffer (25 mM HEPES, pH 7.5, 5 mM MgCl₂, 0.3 M sorbitol) containing the spin trap reagents (50 mM 4‐POBN, 4% ethanol, 50 μM Fe‐EDTA). Infiltrated leaves were placed into the buffer containing the spin trap reagents and illuminated for 1 h with white light (200 μmol photons m⁻² s⁻¹). At the end of the illumination time, the leaves were removed and the EPR signal of the solution was monitored. EPR spectra were recorded at room temperature in a standard quartz flat cell using an ESP‐300 X‐band (9.73 GHz) spectrometer (Bruker). The following parameters were used: microwave frequency 9.73 GHz, modulation frequency 100 kHz, modulation amplitude: 1 G, microwave power: 6.3 mW, receiver gain: 2 × 104, time constant: 40.96 ms; number of scans: 4. The EPR signal sizes were normalised by the total chlorophyll contents of each sample.