True oxygen reduction capacity during photosynthetic electron transfer in thylakoids and intact leaves

Abstract

Reactive oxygen species (ROS) are generated in electron transport processes of living organisms in oxygenic environments. Chloroplasts are plant bioenergetics hubs where imbalances between photosynthetic inputs and outputs drive ROS generation upon changing environmental conditions. Plants have harnessed various site-specific thylakoid membrane ROS products into environmental sensory signals. Our current understanding of ROS production in thylakoids suggests that oxygen (O2) reduction takes place at numerous components of the photosynthetic electron transfer chain (PETC). To refine models of site-specific O2 reduction capacity of various PETC components in isolated thylakoids of Arabidopsis thaliana, we quantified the stoichiometry of oxygen production and consumption reactions associated with hydrogen peroxide (H2O2) accumulation using membrane inlet mass spectrometry and specific inhibitors. Combined with P700 spectroscopy and electron paramagnetic resonance spin trapping, we demonstrate that electron flow to photosystem I (PSI) is essential for H2O2 accumulation during the photosynthetic linear electron transport process. Further leaf disc measurements provided clues that H2O2 from PETC has a potential of increasing mitochondrial respiration and CO2 release. Based on gas exchange analyses in control, site-specific inhibitor-, methyl viologen-, and catalase-treated thylakoids, we provide compelling evidence of no contribution of plastoquinone pool or cytochrome b6f to chloroplastic H2O2 accumulation. The putative production of H2O2 in any PETC location other than PSI is rapidly quenched and therefore cannot function in H2O2 translocation to another cellular location or in signaling.

Figure 1

Schematic description of O₂ reduction, H₂O₂ accumulation, and H₂O₂ decomposition cycle operating in isolated thylakoid membrane samples, highlighting labeled isotope reactions and the targets of specific PETC inhibitors, and catalysts, used in this study. Water oxidation at PSII generates a ¹⁶O₂, represented in blue font, and releases four electrons into the PETC, represented by yellow arrows. These four electrons reduce four artificially enriched ¹⁸O₂, represented in red font. According to the reactions presented at the top right, the superoxide is dismutated into H₂O₂ and finally back to H₂O, reproducing the H₂O originally oxidized but now containing a red, labeled O₂. This final reaction can be catalyzed by externally added catalase (Cat) in the isolated thylakoids, which instantly pushes the cycle to the end. Breaking the complete cycle into partial reactions (Reactions 1–4) provides a stoichiometric gas flux ratio to associate each step of the cycle back to the O₂ production to consumption ratio being measured with MIMS. Reactions 1 and 4 have the same ratio but are distinguished by the O₂ uptake component, occurring only with artificial acceptors. Three well-known inhibitors DCMU, DBMIB, and HgCl₂ are shown at the site of inhibition in electron transport chain that is the PQ-pool reduction at Q_B binding site, the PQ-pool oxidation at Cyt-b₆f and the plastoquinone reduction at Cyt-b₆f. Reaction 5 is a variant of the ratio, predicted from observations that DCMU blocks all water oxidation but samples were still able to consume O₂ in a light dependent manner, suggesting ¹O₂ or organic peroxide formation. PQ, plastoquinone.

Figure 2

Comparison of site-specific inhibitors in blocking electron flow from PSII to PSI. Dark-adapted isolated thylakoids, or intact leaves, were exposed to a saturating pulse (SP) of actinic light. Following this, P700 re-reduction was measured under FR background illumination by firing strong actinic light (10,000 µmol photons m⁻² s⁻¹) pulses for 50 µs (ST) and 50 ms (MT) on (A) isolated thylakoids equivalent to chlorophyll concentration of 80 µg chl mL⁻¹ in measuring buffer containing the uncoupler NH₄Cl (5 mM) and (B) intact leaves. Leaves were infiltrated in darkness using water for the control sample. Inhibitors/modulators were used at 10 µM concentration, except for HgCl₂ used at 2 mg mL⁻¹. Representative curves averaged from minimum three to five biological replicates (n = 3).

Figure 3

Integrated rates of ¹⁶O₂ production and ¹⁸O₂ consumption by isolated thylakoid samples, measured simultaneously at 120 µmol photons m⁻² s⁻¹ with MIMS. Illumination of samples represented by yellow bar with gray representing darkness. Curves A, untreated control; B, untreated control + Cat; C, 10 µM (MV), D, 10 µM MV + Cat (observe increased scale for c and d), E, 10 µM DCMU, F, 10 µM DBMIB and G, 2 mg mL⁻¹ HgCl₂ are the product of averaging minimum three replicates plotted with standard error. H, The ratios of O₂ production to consumption rates were calculated as described in text and plotted as a decimal value to highlight conditions in which H₂O₂ could accumulate, based on the stoichiometry of partial reactions from O₂ reduction to water formation again. All measurements were performed in measurement buffer containing the uncoupler NH₄Cl (5 mM). All curves are an average of minimum three representative replicates (n = 3) plotted with standard error (±SE).

Figure 4

Integrated rates of ¹⁶O₂ production and ¹⁸O₂ consumption by isolated thylakoid samples, measured simultaneously at 900 µmol photons m⁻² s⁻¹ with MIMS. Illumination of samples represented by yellow bar with gray representing darkness. A, untreated control, B, 50 µM DCMU C, 10 µM DBMIB, and D, 2 mg mL⁻¹ HgCl₂. O₂ consumption rates increased more than O₂ production rates in all inhibited samples, compared with measurements at 120 µmol photons m⁻² s⁻¹. F, 50 µM DCMU + Cat suggests ¹O₂ formation as a likely reason for increased O₂ consumption, subsequently this curve was subtracted from both DBMIB and HgCl₂ curves resulting in (G) DBMIB-(DCMU + Cat) and (H) HgCl₂-(DCMU + Cat). E, Plotting O₂ production to O₂ consumption ratios from all curves highlights that subtraction of O₂ consumption associated with peroxidation of lipids, proteins, and membranes by ¹O₂ resulted in the return of O₂ flux ratios that exclude the accumulation of H₂O₂. All curves are an average of minimum three representative replicates (n = 3) plotted with standard error (±SE). All measurements of isolated thylakoids were performed in measurement buffer containing the uncoupler NH₄Cl (5 mM).

Figure 5

Light-induced superoxide formation in isolated thylakoids. Superoxide was measured by spin trapping with DIPPMPO (A) in the dark and (B) after 3 min of illumination with white light (150 μmol photons m⁻² s⁻¹). Typical spectra of DIPPMPO-OOH with hyperfine splitting constants (cis a_P 4.968 mT, a_N 1.314 mT, a_H 1.102 mT; trans a_P 4.95 mT, a_N 1.301 mT, a_H 1.022 mT), DIPPMPO-OH (a_P 4.659 mT, a_N 1.414 mT, a_H 1.339 mT) and DIPPMPO-R (aP 4.59 mT, aN 1. 491 mT, aH 2.22 mT) adducts were measured in the presence of DCMU (Q_B–site inhibitor), HgCl₂ (PC inhibitor), and MV (Catalyst of O₂ reduction at PSI). C, Simulated spectra of the experimental spectrum of control thylakoids from (B), Light upper trace consisting of different proportion of each DIPPMPO-OOH, DIPPMP-OH, and DIPPMPO-R are shown. Thylakoids were isolated from 6-week-old plants, grown under constant light of 120 μmol photons m⁻² s⁻¹ with dark and light cycle of 16/8 h. Thylakoids equivalent to 150 μg chl mL⁻¹ were illuminated with actinic light (150 μmol photons m⁻² s⁻¹) in the presence of 50 mM DIPPMPO, 100 μM desferal, and 50 mM Hepes–NaOH (pH 7.5) with each electron transfer modulator DCMU (10 µM), HgCl₂ (2 mg/150 µg chl), and MV (10 µM). EPR settings were microwave frequency 9.41 GHz, center field 336.2 mT, field sweep 15 mT, microwave power 5 mW, modulation frequency of 100 kHz, modulation width of 0.05 mT, sweep time 60 s. A minimum three to five independently isolated thylakoid samples (n = 3) were used for the final spectra, and five accumulations were recorded from each sample.

Figure 6

The integrated rates versus time of O₂ and CO₂ fluxes from intact leaf discs measured at three different light conditions. Rate versus time plots of (A) control, (B) MV, (D) DBMIB, and (E) DCMU infiltrated leaf discs. Inset shows complete curves and the main figure highlights steady-state rates across the dark and two light intensities GL = 120 (from 300 to 600 s) and HL = 900 (600–720 s) µmol photons m⁻² s⁻¹, respectively. For direct comparison, part C shows CO₂ efflux rates (mitochondrial respiration) from the four treatments. F, O₂ flux ratio associated with Mehler reaction calculated as described in text. All curves average of minimum three representative replicates (n = 3) plotted with standard error (±SE). The apparent mismatch between activation of light and photosynthetic activity is a result of the integration of rates over time required to minimize noise and allowing a focus on steady-state rates.