Oxidative status of Arabidopsis (Arabidopsis thaliana) plants and isolated chloroplast under exposure to excessive light

The study concerns the impact of high light on the photosynthesis parameters and oxidative stress in Arabidopsis plants (Arabidopsis thaliana). In the first stage the impact of 1 and 5 h long high light of 800 or 1000 µM m^-2s^-1 on the photosynthesis process and chlorophyll and 8-OHG levels in RNA was assessed. Based on the results obtained the 1h long exposure to light of 1000 µM m^-2s^-1 was chosen for subsequent evaluation of the levels of oxidation markers (e.g. 8-OHG in RNA, TBARS, ROS) and antioxidant response (e.g. GSH levels, SOD and CAT activity).

Methods:

Photosynthesis parameters were assessed using Fuoropen. Chlorophyll level was measured according to Wellburn 1994 using DMSO as solvent and absorbance measurmetns at λ=665 nm and λ=649 nm.. Chlorophyll content was calculated using following formulas: chlorophyll a = 12,9*(A665) – 3,45*(A649);  chlorophyll b = 21,99*(A649) – 5,32*(A665).

The Level of general reactive oxygen species (ROS) and glutathione (GSH) were measured using Oxidative Stress Combo Assay Tissue Kit (Signosis, EA-7008), ROS by the measn of specific fluorescent dye H2DCFDA and GSH by the means of Ellman’s reagent (DTNB). Lipid peroxidation was assessed by the means of TBARS level measurements. The level of RNA oxidation was assessed by quantification of 8-OHG with two distinct kits – EpiQuik™ 8-OhG RNA Damage Quantification Direct Kit (Epigentek, P-6008) i OxiSelect™ Oxidative RNA Damage ELISA Kit (Cell Biolabs, STA-325).

All measurements were carried out in 2-4 biological repetitions and analyzed statistically with single factor ANOVA test using XL Miner Analysis ToolPak (Microsoft).

The study was funded by Excellence Initiative – Research University (IDUB) in frame of project 140/04/POB2/0005.