E 109 spectrometer

EPR experiments were performed on a Varian E109 spectrometer equipped with a cryogenic system, which allowed for low-temperature data collection. The spectrometer was operated at 9.26 GHz, with a modulation amplitude of 4 G and microwave power of 10 mW, at 70 K. Samples were drawn into quartz tubes and then frozen in liquid nitrogen. The EPR parameters were optimized to avoid line saturation and distortion. The spectrum of the buffer only was used as a baseline and subtracted from all other spectra.

The X-band continuous-wave EPR spectra were recorded at 77 and 300K with a Varian E-109 spectrometer. The frequency of the spectrometer was calibrated with a 2,2-diphenyl-1-picrylhydrazyl (DPPH) standard sample. The weighted portion of copper(II) sulfate pentahydrate (CuSO₄·5H₂O) was used to evaluate the concentration of paramagnetic species. The EPR spectra were simulated in the MATLAB program package with the EasySpin toolbox [70 (link)].

The formation of ROS was measured by EPR spectroscopy using DMPO as a specific superoxide anion spin trap molecule and Tiron as a general spin trap22 (link)23 . Reaction mixture contained chloroplasts equivalent to 50 μg chlorophyll and 10 mM Tiron or 433 mM DMPO whereas in the experiments with MV its respective concentration was 10 mM. Immediately after admission of a specific spin trap, samples were either kept in the dark or illuminated with photosynthetic light of 100 μmol photons m⁻² s⁻¹ for 30 seconds. The experiments were performed in glass capillaries (inner diameter of 1 mm) on an X-band Varian E-109 spectrometer applying the following instrumental set-up: microwave power of 20 mW, modulation amplitude of 0.1 mT, modulation frequency of 100 kHz and scan range of 8 mT (DMPO) and 2.5 mT (Tiron). Data were collected using the supplied software43 at room temperature and analysed by EasySpin software package44 (link). In order to compare the EPR detected radical yield in trol versus WT plants, data related to mutant were normalized with respect to the WT data, which were taken as a referent ones and assumed to represent 100% radical yield. Similarly, when the samples were exposed to MV, the results were quantified with respect to the EPR signal of chloroplasts not exposed to MV, which were taken as a referent ones and assumed to represent 100% radical yield.

The X-band continuous-wave EPR spectra were recorded at 77 and 300 K with a Varian E-109 spectrometer. The frequency of the spectrometer was calibrated with a 2,2-diphenyl-1-picrylhydrazyl (DPPH) standard sample. The weighted portion of copper(II) sulfate pentahydrate (CuSO₄·5H₂O) was used to evaluate the concentration of paramagnetic species. The EPR spectra were simulated in the MATLAB program package with the EasySpin toolbox [39 (link)].