Formation of α-tocopherol hydroperoxide and α-tocopheroxyl radical: relevance for photooxidative stress in Arabidopsis

Tocopherols, lipid-soluble antioxidants play a crucial role in the antioxidant defense system in higher plants. The antioxidant function of α-tocopherol has been widely studied; however, experimental data on the formation of its oxidation products is missing. In this study, we attempt to provide spectroscopic evidence on the detection of oxidation products of α-tocopherol formed by its interaction with singlet oxygen and lipid peroxyl radical. Singlet oxygen was formed using photosensitizer rose bengal and thylakoid membranes isolated from Arabidopsis thaliana. Singlet oxygen reacts with polyunsaturated fatty acid forming lipid hydroperoxide which is oxidized by ferric iron to lipid peroxyl radical. The addition of singlet oxygen to double bond carbon on the chromanol head of α-tocopherol forms α-tocopherol hydroperoxide detected using fluorescent probe swallow-tailed perylene derivative. The decomposition of α-tocopherol hydroperoxide forms α-tocopherol quinone. The hydrogen abstraction from α-tocopherol by lipid peroxyl radical forms α-tocopheroxyl radical detected by electron paramagnetic resonance. Quantification of lipid and protein hydroperoxide from the wild type and tocopherol deficient (vte1) mutant Arabidopsis leaves using a colorimetric ferrous oxidation-xylenol orange assay reveals that α-tocopherol prevents formation of both lipid and protein hydroperoxides at high light. Identification of oxidation products of α-tocopherol might contribute to a better understanding of the protective role of α-tocopherol in the prevention of oxidative damage in higher plants at high light.

Thylakoid membrane preparation. Thylakoid membranes were isolated from high light exposed plants using the protocol developed by Casazza et al. 25 . Harvesting of rosette leaves (0.3-0.5 g) from plants was done followed by floating them on ice-cold water for 10-20 min in the dark and then blotted. Leaves were rapidly homogenized in 10-20 ml of grinding buffer comprising EGTA (5 mM), EDTA (5 mM), MgCl 2 (5 mM), NaHCO 3 (10 mM), sorbitol (0.4 M) and tricine/NaOH (20 mM, pH 8.4) and 0.5% (w/v) fatty acid-free BSA was added just before the grinding. Homogenized suspension was filtered through 2 layers of cheesecloth by applying a gentle hand pressure to increase the final thylakoid yield. The filtrate was centrifuged at 2600 g for 3 min at 4 °C, followed by re-suspending the pellet in 10-20 ml of resuspension buffer containing EDTA (2.5 mM), HEPES (20 mM, pH 7.6) MgCl 2 (5 mM), NaHCO 3 (10 mM), sorbitol (0.3 M) and 0.5% (w/v) fatty acid-free BSA. Centrifugation was done at 2600 g for 3 min at 4 °C and the pellet was washed again in re-suspension buffer without adding fatty acid-free BSA and then resuspended in 10-20 ml of hypotonic buffer containing EDTA (2.5 mM), MgCl 2 (5 mM), NaHCO 3 (10 mM), HEPES (20 mM, pH 7.6) and 0.5% (w/v) fatty acid-free BSA. Thylakoid membranes were collected by centrifugation at 2600 g for 3 min at 4 °C. Finally, the pellet was suspended in a small volume (0.5-1 ml) of resuspension buffer and was stored at -80 °C in the dark until use. The chlorophyll concentrations from thylakoid preparations were calculated from the absorbance at 645 and 663 nm of 80% (v/v) acetone extract, according to Arnon 26 . Determination of α-tocopherol and α-tocopherol quinone by HPLC. The amount of α-TOH and α-TQ was assessed by the reverse-phase HPLC analysis using postcolumn reduction with platinum following the protocol described in Nowicka and Kruk 27 . To avoid the auto-oxidation of α-TOH and α-TQ standard, we stored standard at -80 °C in several concentrations. To avoid the auto-oxidation of α-TOH and α-TQ extracted from leaves, we performed the liquid extraction of α-TOH and α-TQ in chilled methanol. We considered the preanalytical methods mentioned in Giusepponi et al. 28 . α-TOH (30 µM) in the presence of rose bengal (5 µM ) and from thylakoid membranes (750 µg Chl ml −1 ) was extracted in methanol by vortexing (5 min) and centrifuged at 2000 g for 60 s at 4 °C, the supernatant was transferred to HPLC vial using a syringe with needle. Minimum of three independent biological replicates were measured to enable an assessment of significance. Isocratic analysis (0.8 ml min −1 at 25 °C) was done using methanol as mobile phase and a LiChrospher 100 RP-18 column (5 µm) LiChroCART 250-4 (Merck, Darmstadt, Germany). Alliance e 2695 HPLC System (Waters Corporation, Milford MA, USA) equipped with a 2998 Photodiode Array (PDA) and a 2475 Fluorescence (FLR) detectors were used. Operation and data processing was performed by Empower 3 Chromatography Data Software (Waters Corporation, Milford MA, USA) (https ://www.water s.com/water s/en_US/Empow er-3-Chrom atogr aphy-Data-Softw are). For determination of α-TOH, fluorescence detection was used (λ ex = 290 nm, λ em = 330 nm). To quantify α-TOH and α-TQ, the calibration curve established in our lab by plotting the peak area at the wavelength for various concentrations of standards was used. α-TOH and α-TQ standards were obtained from Sigma Aldrich GmbH (Germany).

Quantification of lipid and protein hydroperoxides by absorption spectroscopy.
To determine LOOH and protein hydroperoxides (POOH) concentration in high light illuminated leaves, lipid and protein extraction was done by following the method of Grintzalis 31 . Leaves harvested from high light illuminated plants were weighed, approximately equal fresh weight of leaves was homogenized in 2-5 ml of 10 mM inorganic phosphate buffer containing 0.5 mM butylated hydroxytoluene (BHT), homogenate was centrifuged at 20,000 g for 10 min at 4 °C and then the filtrate was vigorously vortexed with 2-5 ml of chloroform (CHCl 3 ): methanol (CH 3 OH) (2:1) for the lipid extraction. After vigorous vortex, 100% trichloroacetic acid (TCA) (volume of 100% TCA should be 10% of phosphate buffer used for homogenization) was added. The mixture was vortexed for 30-60 s and incubated in ice-cold water for 20 min followed by centrifugation at 20,000g for 10 min at 4 °C. After centrifugation, three separate layers (top aqueous layer, middle protein disc and the lower layer containing lipids) were visible. Lower lipid layer was collected in a fresh Eppendorf tube, dried under the nitrogen stream and used for FOX assay. Protein discs were washed with 10% TCA using ultrasonic homogenizer, homogenized protein discs were centrifuged at 20,000g for 10 min at 4 °C and the pellet was dissolved in urea (8 M) and used for FOX assay. FOX assay was performed in three biological replicates to confirm the significance of measurements. FOX reagent was prepared fresh by dissolving 15

Consumption of α-tocopherol detected by HPLC.
To monitor the consumption of α-TOH by its oxidation during 1 O 2 quenching and LOO • scavenging, the amount of α-TOH was determined by the reversephase HPLC analysis using a fluorescence detector. Figure 1 shows the chromatograms of α-TOH added to rose bengal (Fig. 1A) and α-TOH extracted from thylakoid membranes (Fig. 1B). Under the chromatographic conditions used in this study, the observed chromatograms show the peak corresponding to α-TOH at retention time 12.7 min in both rose bengal (Fig. 1A, dark trace) and thylakoid membranes (Fig. 1B, dark trace). When rose bengal and thylakoid membranes were illuminated, suppression of the peak at retention time 12.7 min was observed ( Fig. 1A and B, light trace). The concentration of α-TOH in rose bengal (Fig. 1C) and thylakoid membranes (Fig. 1D) in dark was 6.12 ± 0.18 nmol ml −1 and 0.55 ± 0.00 nmol ml −1 , where it decreases to 0.63 ± 0.19 nmol ml −1 and 0.06 ± 0.03 nmol ml −1 in light, respectively. Rose bengal-photosensitized 1 O 2 formation caused complete consumption of α-TOH due to 1 O 2 quenching, whereas α-TOH consumption in thylakoid membranes was due to 1 O 2 quenching and LOO • scavenging.
Singlet oxygen quenching by α-tocopherol monitored by EPR spectroscopy. Singlet oxygen quenching by α-TOH was studied by EPR spectroscopy using TMPD as a spin probe. Oxidation of diamagnetic TMPD by 1 O 2 forms paramagnetic TEMPONE detected by EPR spectroscopy. Singlet oxygen was generated either by photosensitization of rose bengal or illumination of thylakoid membranes isolated from Arabidopsis. Addition of TMPD spin probe to rose bengal or thylakoid membranes in the dark did not result in the appearance of TEMPONE EPR spectra ( Fig. 2A and B, control trace and Fig. 2C and D, control bar), whereas photosensitization of rose bengal or illumination of thylakoid membranes resulted in the formation of TEMPONE EPR signal ( Fig. 2A   www.nature.com/scientificreports/ illumination of thylakoid membranes was performed in the presence of α-TOH, TEMPONE EPR signal was significantly suppressed ( Fig. 2A and B, 1 O 2 + α-TOH trace and Fig. 2C and D, 1 O 2 + α-TOH bar). These observations indicate that α-TOH serves as an efficient quencher of 1 O 2 generated from the photosensitization of rose bengal or illumination of Arabidopsis thylakoid membranes.
Formation of α-tocopherol hydroperoxide detected by fluorescence spectroscopy. To monitor the formation of α-TOOH after oxidation of α-TOH by 1 O 2 , fluorescent probe swallow-tailed perylene derivative (SPY-LHP) was used. In this reaction, the oxidation of non-fluorescent SPY-LHP by α-TOOH forms fluorescent oxidized derivative (SPY-LHPox) which provides fluorescence in the green region of the spectrum. Fluorescence spectrum of SPY-LHPox shows fluorescence maximum at 538 nm and 575 nm (Fig. 3A, lower dashed trace) as previously shown by Soh et al. 30 . When SPY-LHPox fluorescence spectrum was measured in the presence of rose bengal and α-TOH in dark, no change was seen in fluorescence at 538 nm whereas, fluorescence at 575 nm was increased due to fluorescence of rose bengal (Fig. 3A, control trace). Rose bengal-photosensitized 1 O 2 formation caused a significant enhancement in SPY-LHPox fluorescence at 538 nm due to α-TOOH formation (Fig. 3A, α-TOOH trace and Fig. 3C, α-TOOH bar). It cannot be excluded that the formation of rose bengal hydroperoxide contributes to the enhancement in SPY-LHPox fluorescence. To confirm that α-TOOH is reduced to α-TOH by ascorbate, the effect of sodium ascorbate on α-TOOH formation was studied. When sodium ascorbate was added to α-TOOH formed by the photosensitized oxidation of α-TOH, the SPY-LHPox fluorescence was decreased (Fig. 3A, α-TOOH + Asc trace and Fig. 3C, α-TOOH + Asc bar). These observations reveal that oxidation of α-TOH by 1 O 2 forms α-TOOH which is reduced back to α-TOH by ascorbate. When SPY-LHPox fluorescence spectrum was measured in the thylakoid membranes in dark, SPY-LHPox fluorescence was low due to fluorescence of only SPY-LHP (Fig. 3B, control trace and Fig. 3D, control bar). When thyla- www.nature.com/scientificreports/ koid membranes were illuminated in the presence of SPY-LHP, increase in the SPY-LHPox fluorescence was observed (Fig. 3B, ROOH trace and Fig. 3D, ROOH bar). As thylakoid membranes are abundant in lipid and protein, SPY-LHPox fluorescence may be due to the formation of organic hydroperoxides comprising LOOH and POOH. The addition of sodium ascorbate to thylakoid membranes caused a decrease in SPY-LHPox fluorescence (Fig. 3B, ROOH + Asc trace and Fig. 3D, ROOH + Asc bar) confirming that α-TOOH is reduced to α-TOH by ascorbate and thus it prevents the formation of ROOH. These results indicate that α-TOH prevents oxidation of lipids and proteins by 1 O 2 quenching. It is proposed here that detection of α-TOOH from thylakoid membranes might be feasible after separation of α-TOOH from other organic hydroperoxides (LOOH, POOH) and identification by 1 H-NMR and mass spectrometry 32 .

Formation of α-tocopherol quinone detected by HPLC.
To study the formation of α-TQ by decomposition of α-TOOH, the amount of α-TQ was determined by the reverse-phase HPLC analysis using postcol- with green light obtained using band-pass interference filter (λ = 560 nm, HBW = 10 nm). In B, SPY-LHPox fluorescence spectra were obtained by the illumination of thylakoid membranes (20 µg Chl ml −1 ) with red light using a long-pass edge interference filter (λ > 600 nm). In C, Quantification of the α-TOOH generated by photosensitization of rose bengal and the effect of sodium ascorbate. In D, Quantification of the ROOH generated by photosensitization of chlorophyll and the effect of α-TOH regeneration by sodium ascorbate. Illumination of rose bengal and thylakoid membranes with SPY-LHP (2.5 µM) was done with green and red light (1000 µmol photons m −2 s −1 ) for 5 min and 15 min in the absence or presence of α-TOH (20 µM) and sodium ascorbate (500 µM), respectively. The SPY-LHPox fluorescence intensity at 538 nm was used to quantify the rose bengal-photosensitized formation of α-TOOH and chlorophyll photosensitized formation of ROOH. Each data point represents the mean ± SD of biological replicates (n = 3).

Formation of lipid and protein hydroperoxides by absorption spectroscopy. As SPY-LHP reacts
with several types of organic hydroperoxides (LOOH, POOH, α-TOOH), LOOH and POOH were isolated from WT and vte1 Arabidopsis leaves and quantified using a colorimetric ferrous oxidation-xylenol orange (FOX) assay. In this assay, Fe 3+ formed by the oxidation of Fe 2+ by ROOH interacts with xylenol orange (XO) in acidic environments forming an XO-Fe complex with the maximum absorption at 560 nm. The concentration of LOOH (Fig. 5A) and POOH (Fig. 5B) formed in high light illuminated WT and vte1 Arabidopsis leaves were in the range of several tens of nanomoles. Relatively higher LOOH formation in vte1 suggests that α-TOH prevents LOOH formation in Arabidopsis leaves at high light.

Discussion
The antioxidant mechanism of α-TOH was previously examined in many studies 14,[33][34][35] . In the current study, we have shown that consumption of α-TOH caused either by photosensitization of rose bengal (Fig. 1A) or illumination of thylakoid membranes (Fig. 1B) is associated with formation of α-TOH oxidation products: (1) oxidation of α-TOH by 1 O 2 forms α-TOOH and (2) oxidation of α-TOH by LOO • forms α-TO • . When other antioxidants are present such as ascorbate, oxidation products of α-TOH (α-TOOH and α-TO • ) are recycled back to α-TOH. Since ascorbate is present in a higher amount than α-TOH, it forms a large reservoir of antioxidant, which could effectively maintain α-TOH restoration. As the most important structural feature of α-TOH in antioxidant activity is chromanol head, oxidation reactions underlying the interaction of 1 O 2 and LOO • with electron-rich double bonds in chromanol head are discussed below. Arabidopsis quantified by colorimetric ferrous oxidation-xylenol orange (FOX) assay. The concentration of LOOH and POOH was established from the calibration curve obtained using hydrogen peroxide. Each data point represents the mean ± SD of biological replicates (n = 3). A significant difference between high light-exposed WT and vte1 Arabidopsis plant is indicated by the asterisk ** (Student's test p < 0.001).
Scientific Reports | (2020) 10:19646 | https://doi.org/10.1038/s41598-020-75634-0 www.nature.com/scientificreports/ Oxidation of α-tocopherol by singlet oxygen forms α-tocopherol hydroperoxide. It was formerly shown that the methylene blue-photosensitized oxidation of α-TOH results in the formation of α-TOOH in model systems 36,37 . Detection of α-TOOH using fluorescent probe SPY-LHP showed that 1 O 2 photosensitized by rose bengal caused significant formation of α-TOOH (Fig. 3A). It was previously proposed that reaction of α-TOH with 1 O 2 occurs via ene reaction 36 . In agreement with this, we propose here that oxidation of α-TOH by 1  It was formerly shown that α-TOOH formed by the methylene blue-photosensitized oxidation of α-TOH is reduced to α-TOH by ascorbate 37 . Our results of α-TOOH formed by the rose bengal photosensitized oxidation of α-TOH and its reduction to α-TOH by ascorbate ( Fig. 3A and C) agrees with the previous reports. Interestingly, the observation that α-TOOH was not fully reduced to α-TOH reveals that α-TOOH decomposes to α-TQ.
Oxidation of α-tocopherol by lipid peroxyl radical forms α-tocopheroxyl radical. It is well established that major antioxidant function of α-TOH is LOO • scavenging 38 . Scavenging of LOO • by α-TOH is maintained because hydrogen transfer from α-TOH to LOO • is faster than hydrogen transfer from neighboring fatty acid 39,40 . Due to the stereoelectronic features of α-TOH, the stability of α-TO • formed after the hydrogen transfer from α-TOH to LOO • is relatively high. The reaction between α-TOH and LOO • occurs either through concerted hydrogen transfer or via sequential electron transfer followed by proton transfer to form LOOH and α-TO • (Fig. 4A and B, α-TO • trace). The α-TO • can either be reduced back to α-TOH by another cellular reductant such as ascorbate forming Asc •− (Fig. 4A and B, Asc •− trace) or react with another LOO • forming nonradical products. Tocopherol dimers and trimers may be formed during LOO • scavenging as minor products 41  -e -, -H + α-Tocopherol α-Tocopheroxyl radical Continuous antioxidant activity provided by α-TOH depends on reductive regeneration of α-TOH from α-TO • by ascorbate 42 . It is well established that α-TO • is reduced by ascorbate to α-TOH while Asc •− is formed [43][44][45][46][47] . Due to continuous regeneration of α-TOH by ascorbate, LOO • scavenging by α-TOH allows suppression of lipid peroxidation at concentrations typically as low as one molecule of α-TOH per thousand phospholipids 34,38,48-52 . Relevance for photooxidative stress in Arabidopsis. It was previously demonstrated that herbicide pyrazolynate mediated-inhibition of α-TOH biosynthesis in Chlamydomonas cells under high light stress caused PS II inactivation 53 . Using WT and vte1 Arabidopsis exposed to high light at low temperature, α-TOH was shown to protect lipids from the photooxidative damage 54,55 . Our observation that formation of LOOH (Fig. 5A) and POOH (Fig. 5B) in WT was lower compared to vte1 Arabidopsis reveals that α-TOH prevent formation of both LOOH and POOH.

Conclusion
The aim of this study was to contribute to the understanding of in vitro antioxidant mechanisms of α-TOH against photooxidative stress. Detail description of α-TOOH formation by 1 O 2 chemical quenching and α-TO • formation by LOO • /ROO • scavenging might help to elucidate antioxidant activity of α-TOH in Arabidopsis plants. Mechanism of the cellular antioxidant defense plays a crucial role in regulating the levels of 1 O 2 and LOO • /ROO • in plants when exposed to a variety of environmental stresses.