TROL-FNR interaction reveals alternative pathways of electron partitioning in photosynthesis

In photosynthesis, final electron transfer from ferredoxin to NADP+ is accomplished by the flavo enzyme ferredoxin:NADP+ oxidoreductase (FNR). FNR is recruited to thylakoid membranes via integral membrane thylakoid rhodanase-like protein TROL. We address the fate of electrons downstream of photosystem I when TROL is absent. We have employed electron paramagnetic resonance (EPR) spectroscopy to study free radical formation and electron partitioning in TROL-depleted chloroplasts. DMPO was used to detect superoxide anion (O2.−) formation, while the generation of other free radicals was monitored by Tiron. Chloroplasts from trol plants pre-acclimated to different light conditions consistently exhibited diminished O2.− accumulation. Generation of other radical forms was elevated in trol chloroplasts in all tested conditions, except for the plants pre-acclimated to high-light. Remarkably, dark- and growth light-acclimated trol chloroplasts were resilient to O2.− generation induced by methyl-viologen. We propose that the dynamic binding and release of FNR from TROL can control the flow of photosynthetic electrons prior to activation of the pseudo-cyclic electron transfer pathway.

determined according to Giannopolities and Ries (R2). The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.5), 13 mM methionine, 75 μM NBT, 0.1 mM EDTA, 2 μM riboflavin, and enzyme extract. The mixture was illuminated (~120 μmol photons m -2 s -1 ) at room temperature for 10 minutes and the absorbance was read at 560 nm. The same one, without illumination, was used as blank. One unit of activity was defined as the amount of enzyme required to cause 50% inhibition of reduction of nitroblue tetrazolium (NBT), compared to the one without enzyme. The extraction for GR activity were done in 100 mM ice cold potassium phosphate buffer (pH 7.0) containing 5 mM sodium ascorbate and 1 mM EDTA. The reaction mixture contained 100 mM potassium phosphate buffer (pH 7.5), 1 mM EDTA, 2 μM GSSG, 2 μM NADPH and enzyme extract. GR activity was assayed by following the decrease of absorbance caused by the oxidation of NADPH at 340 nm, according to Bitensky (R3).
Determination of TBARS, H2O2 levels and protein carbonyls. The concentration of thiobarbituric acid-reacting substances (TBARS), as a measure of lipid peroxidation, was monitored as described by Verma and Dubey (R4). Powdered fresh samples were extracted with 1 ml 0.1% (w/v) trichloroacetic acid (TCA).
After centrifugation at 6000xg for 5 min, 0.5 ml of the supernatant was added to 1 ml 0.5% (w/v) thiobarbituric acid (TBA) in 20% TCA. The mixture was heated at 95°C for 30 min and cooled in an ice-bath. After centrifugation (18,000xg, 15 min, 4°C) the absorbance of the supernatant was measured at 532 nm (correction was done by subtracting the absorbance at 600 nm for unspecific turbidity), and the concentration of lipid peroxidation products was expressed as total TBARS in terms of nmol g -1 dry weight (extinction coefficient 155 mM -1 cm -1 ). For the determination of H2O2 levels powdered leaf tissue was extracted with ice-cold acetone and centrifuged. Titanium sulphate and NH4OH were added into extracts. The resulting pellet was dissolved with 2 M H2SO4 and absorbance was read at 415 nm. The concentration of H2O2 was calculated using an extinction coefficient of 1.878 mM -1 cm -1 , according to Mukherjee and Choudhuri (R5). Powdered samples for determination of carbonyl groups were extracted with 100 mM potassium phosphate buffer (pH 7.5). Homogenates were assayed according to the method of Levine et al. (R6) in which carbonyl groups on protein side were derivatised to 2,4-dinitrophenylhydrazone by reaction with 2,4-dinitrophenylhydrazine (DNPH) dissolved in HCl. The protein contents were determined on HCl-blank pellets and absorbance was read at 280 nm. The content of carbonyl groups was calculated using a bovine serum albumin (BSA) standard curve (BSA is dissolved in urea HCl).
Determination of AA concentration. As a non-enzymatic antioxidant, AA was determined according to Mukherjee and Choudhuri (R5). Powdered fresh samples were extracted in 6% TCA. After centrifugation, 2% DNPH in 50% H2SO4 and 10% thiourea in 70% ethanol were added into the supernatant. The mixture was boiled in water bath for 15 min, cooled at RT and centrifuged. Resulting precipitate was dissolved in 80% H2SO4 and the absorbance was read at 530 nm. An extinction coefficient of 226.2 mM -1 cm -1 was used for calculation.  Table S1. Table S1. Mann-Whitney U test of measurements of enzymatic and non-enzymatic components of antioxidative stress response in Arabidopsis WT and trol plants grown under the growth light (80 µmol photons m -2 s -1 ). Lipid peroxidation level; hydrogen peroxide (H2O2); catalase (CAT); ascorbic acid concentration (AA), superoxide dismutase (SOD); protein carbonyls; and glutathione reductase (GR) were detected and quantified in WT and trol plant leaves. All data were analysed by Mann-Whitney U test. Significant differences between the values are indicated by asterisk (significantly higher value, p<0.01). Supplemental Figure S2. The EPR study of DMPO-OOH spin adduct and Tiron semiquinone radical formation in chloroplasts isolated from Arabidopsis trol plants acclimated to different light conditions (dark, GL or HL). Chloroplasts were illuminated with photosynthetic light of 100 µmol photons m -2 s -1 for 30 seconds (white columns) or kept in dark for the equivalent time period (black columns). (a) The superoxide anion production from chloroplasts exposed to MV normalized with respect to the samples not exposed to MV that are taken as a reference for 100% superoxide radical production;

Supplemental
(b) Data from chloroplasts exposed to MV normalized with respect to the samples not exposed to MV assumed to represent 100% radical yield. Data (mean ± SE) from three independent measurements were analysed by one-way ANOVA and LSD post-hoc test. Data labelled with the same lower-case letters are not significantly different. Different lower-case letters point to the experimental data which are significantly different (p<0.05).