Role of plastoglobules in metabolite repair in the tocopherol redox cycle

Plants are exposed to ever changing light environments and continuously forced to adapt. Excessive light intensity leads to the production of reactive oxygen species that can have deleterious effects on photosystems and thylakoid membranes. To limit damage, plants increase the production of membrane soluble antioxidants such as tocopherols. Here, untargeted lipidomics after high light treatment showed that among hundreds of lipid compounds alpha-tocopherol is the most strongly induced, underscoring its importance as an antioxidant. As part of the antioxidant mechanism, α-tocopherol undergoes a redox cycle involving oxidative opening of the chromanol ring. The only enzyme currently known to participate in the cycle is tocopherol cyclase (VTE1, At4g32770), that re-introduces the chromanol ring of α-tocopherol. By mutant analysis, we identified the NAD(P)H-dependent quinone oxidoreductase (NDC1, At5g08740) as a second enzyme implicated in this cycle. NDC1 presumably acts through the reduction of quinone intermediates preceding cyclization by VTE1. Exposure to high light also triggered far-ranging changes in prenylquinone composition that we dissect herein using null mutants and lines overexpressing the VTE1 and NDC1 enzymes.


INTRODUCTION
In their natural environment, plants are exposed to many kinds of stress, such as heat, drought and high light (Suzuki et al., 2012) especially during summer or due to anthropogenic activities including herbicides, air pollutants and acid rain (Lichtenthaler, 1998). For plants growing under field conditions, occasional exposure to high or even excessive light intensities is normal but has the potential to damage the photosynthetic apparatus. Exposure to high light (HL) generates reactive oxygen species (ROS) in chloroplasts, principally singlet oxygen in PSII (Krieger-Liszkay, 2005) and superoxide in PSI (Asada, 1999). To protect membrane lipids from photooxidation and PSII from photoinhibition, higher plants have developed a variety of adaptive strategies. In addition to modifications of pigment composition (Lichtenthaler et al., 2007) and chloroplast ultrastructure Brehelin et al., 2007), plants synthetize various lipid and water soluble antioxidants such as tocopherol (vitamin E) and ascorbate, respectively (Delong and Steffen, 1997;Noctor and Foyer, 1998;Smirnoff, 2000;Smirnoff and Wheeler, 2000;Sattler et al., 2003;Havaux et al., 2005;Van Breusegem et al., 2008). Storage and metabolism of tocopherol but also phylloquinone (Vit K), plastoquinone (PQ) and its derivative plastochromanol (PC8) (Lohmann et al., 2006;Vidi et al., 2006;Szymanska and Kruk, 2010b;Zbierzak et al., 2010;Eugeni Piller et al., 2012) in part take place at chloroplast lipid droplets (plastoglobules; PG) implicating them in light stress responses. PG are attached to the thylakoid membrane by the shared outer lipid leaflet. This arrangement leads to a conduit that may allow the diffusion of lipid molecules between the two compartments . At least, two metabolic enzymes involved in prenylquinone pathways are located at PG: the tocopherol cyclase VTE1 and NDC1 Ytterberg et al., 2006;Eugeni Piller et al., 2011;Lundquist et al., 2012). In addition, two kinases ABC1K3 and ABC1K1/PGR6 have been implicated in the regulation of PC8 production as well as α-tocopherol overaccumulation under HL. They may function via phosphorylation of VTE1 (Lundquist et al., 2012;Martinis et al., 2013Martinis et al., , 2014. Under HL stress, the synthesis of tocopherol is enhanced suggesting that this molecule exerts an essential role as lipid antioxidant (Munne-Bosch, 2005;Eugeni Piller et al., 2012). It has been shown that tocopherol is important in the maintenance of PSII function (Porfirova et al., 2002;Havaux et al., 2005). A large proportion of total plastid tocopherol is accumulated in the PG core which enlarges during oxidative stress Brehelin et al., 2007).
In response to HL stress, plants accumulate tocopherol oxidation products. In contrast to animal membranes, only one such product has been reported to accumulate in plants, namely α-tocopherol quinol (α-TQH 2 ) ( Figure 1B) Mene-Saffrane and Dellapenna, 2010). A part from being a product of tocopherol oxidation, several functions for α-TQH 2 have been proposed: dissipation of excess energy, protection of PSII against photoinhibition (Kruk et al., 2000(Kruk et al., , 2003Munne-Bosch, 2005) as well as a strong antioxidant activity (Kruk and Trebst, 2008;Nowicka and Kruk, 2010).
Recent studies demonstrate the existence of a plastidbased mechanism for a tocopherol redox cycle Mene-Saffrane and Dellapenna, 2010;Eugeni Piller et al., 2012). In the first step of this recycling pathway, an α-tocopherol radical is formed by α-tocopherol oxidation via a lipid peroxy radical (LOO − ). This compound is then oxidized by a second lipid peroxy radical to form α-tocopherol quinone (α-TQ) that is successively reduced to give α-TQH 2 . A yet unidentified plastid-dehydratase activity converts α-TQH 2 to trimethylbenzoquinone (TMPBQ) (or TMPBQH 2 ), which is then cyclized by VTE1 leading to the regeneration of α-tocopherol and completion of the cycle.
In the present study, we used ultra-high pressure liquid chromatography-mass spectrometry to analyse the composition of prenylquinones that play a fundamental role in light stress response in a variety of genetic backgrounds. We report that the PG-localized NAD(P)H-dependent quinone oxidoreductase NDC1 participates in the tocopherol redox cycle. NDC1 most likely functions by reducing α-TQ to α-TQH 2 . This hypothesis is supported by the implication of NDC1 in the analogous reduction of PQ to PQH 2 in PG under HL stress .

OVEREXPRESSION OF NDC1 IN A. thaliana LEAVES
Plants overexpressing NDC1-YFP under the 35S promoter were obtained using the Gateway recombination technology (Invitrogen): The NDC1 coding sequence was introduced into a donor vector pDONR™221, and subsequently transferred into an appropriate destination vector, the pEarlyGate101-YFP binary vector, resulting in pEarlyGate101-NDC1-YFP. pEarlyGate101-NDC1-YFP was transferred into Arabidopsis WT plants using the floral dip method (Clough and Bent, 1998). Transformed plants were selected for BASTA resistance and confirmed by segregation analysis.

WESTERN BLOT ANALYSIS
Total protein was isolated from Arabidopsis leaves according to Rensink et al. (1998) and concentrated by chloroform-methanol precipitation (Wessel and Flugge, 1984). Twenty μg of protein were separated by SDS-PAGE and blotted onto nitrocellulose membrane for immunodetection. Immunodetection was carried out using anti-NDC1 serum at 1/1000 dilution in 5% fat free milk powder/TBS .

CONFOCAL MICROSCOPY
Protoplasts were released from plants overexpressing NDC1-YFP by overnight digestion with macerozyme (0.25%, Serva) and reduced cellulase (1%, Serva) in a solution containing 400 mM Mannitol, 5 mM MES and 8 mM CaCl 2 . Protoplasts were filtered and loaded on a sucrose gradient (21 and 42%) and centrifuged for 10 min at 50 × g. Intact protoplasts were resuspended and fluorescence was monitored with a Leica TCS SP5 confocal microscope using the appropriate parameters for YFP (514-nm laser lines, 520-588-nm detection windows).

PRENYLLIPID EXTRACTION FROM WHOLE PLANTS OR PG FRACTIONS AND LIPIDOMICS PROFILING
Prenylquinones were extracted from whole plants using an established method (Martinis et al., 2011). Leaves were ground in liquid nitrogen in a mortar. 100 mg were re-suspended in 500 μl of tetrahydrofuran (THF, analytical grade, Normapur). Glass beads (1 mm) were added and samples homogenized at 30 Hz, 3 min (Retsch MM 300). After centrifugation, 200 μl were transferred to a suitable HPLC vial.
To measure prenylquinones contained in PG, intact chloroplasts were isolated by centrifugation on a Percoll gradient. Subsequently, PG were separated from thylakoid membranes by flotation on a sucrose gradient as described in Besagni et al. (2011). Four hundred μl of PG and thylakoid fractions were added to 600 μl of water and extracted three times with an equal volume of ethylacetate. Organic phases were pooled, evaporated and pellets were dissolved in 100 μl of THF/water (85/15 v/v) and the solution transferred to an appropriate HPLC vial (Kessler and Glauser, 2014).

DATA PRE-PROCESSING AND STATISTICAL ANALYSIS
For comparison of metabolic profiles, raw spectrometric data were processed using Markerlynx XS™ (Waters) which performs automatic peak detection and deconvolution in each chromatogram. The parameters were as follows: initial and final retention times 0.5-3.0 min, mass range m/z 300-1200 Da, mass tolerance 0.02 Da, retention time window 0.10 min, automatic peak with detection, automatic measurement of peak-to-peak baseline noise, intensity threshold 400 counts, no smoothing, noise elimination level disabled, deisotope filtering function applied. Peak areas for individual variables were normalized to the total integrated area per sample. Variables were Pareto-scaled before applying principal component analysis (PCA).

STATISTICAL TESTS
Multivariate statistical analysis of lipid profiles was performed using EZinfo (Umetrics). Univariate Analyses were performed using the software SigmaPlot version 12.0. Data were first analyzed using Shapiro-Wilk to determine whether data were normally distributed. When data passed the test, the Student's test (t-test) was applied to evaluate statistically significant difference between values (p < 0.05). For non-normally distributed data, the Mann-Whitney U-test was used.

CHARACTERIZATION OF NDC1 OVEREXPRESSING PLANTS
To improve our understanding of prenylquinone biosynthesis and metabolic regulation, we used Arabidopsis wild type (WT) plants, vte1 and ndc1 mutants as well as 35S:VTE1-YFP and 35S:NDC1-YFP overexpressing lines exposed to either moderate light conditions or HL stress in lipidomics studies. With the exception of 35S:NDC1-YFP the different lines were characterized previously, including two independent homozygous ndc1 mutant lines SALK_024063 and GABI_614F03 ) that showed no differences in phenotype or prenylquinone composition. The transgenic plants overexpressing NDC1 fused to yellow fluorescent protein (YFP) under the 35S promoter (35S:NDC1-YFP) were engineered for this study. The expression of NDC1-YFP was verified by Western blotting using an antibody against NDC1 (Figure 2A). 35S:NDC1-YFP plants but neither wild type nor ndc1 gave a band at around 80 kDa corresponding to the predicted mass of the fusion protein. Endogenous NDC1 protein (57 kDa) was not detected in WT and overexpressing plants due to the low amount of total protein (20 μg) loaded (100 μg are necessary; Eugeni Piller et al., 2011). The 35S:NDC1-YFP plants had no apparent phenotype.
Protoplasts isolated from 35S:NDC1-YFP plants were analyzed by confocal microscopy and gave punctate fluorescence inside the chloroplasts which is in agreement with the PG localization of NDC1 Ytterberg et al., 2006;Eugeni Piller et al., 2011;Lundquist et al., 2012) (Figure 2B). Moreover, the 35S:NDC1-YFP construct was previously tested by transient expression in Arabidopsis WT protoplasts in the presence of the neutral lipid dye Nile Red which stains PG. The NDC1-YFP and Nile Red signals colocalized by confocal microscopy .

NDC1 AND VTE1, TWO PLASTOGLOBULE ENZYMES INVOLVED IN PRENYLQUINONE METABOLISM
To understand the dynamics of prenylquinone synthesis under changing light conditions, we first analyzed the global lipid The data obtained, using the UHPLC-QTOFMS-based method, were subjected to multivariate analysis. Using this method more than 500 different compounds were detected, not all of which could be identified (Supplementary Table 1). To investigate the difference in lipid contents, a principal component analysis (PCA) model was established from the data sets. PCA identifies and ranks major sources of variance and allows clustering of samples based on similarities and differences in the measured parameters.
Under moderate light conditions, PCA showed the separation of five distinct groups characteristic for each of the genotypes tested in triplicate ( Figure 3A). PCA loadings revealed that prenylquinones mostly contributed to the separation of these groups ( Figure 3B). Most of the other lipids extracted were near the origin, suggesting that their contribution to metabolic difference was negligible.
The separation of ndc1 from the other genotypes was mainly based on the presence of the 2-phytyl-1,4-naphthoquinone, the de-methyl precursor of phylloquinone . Interestingly, an accumulation of PQ was also observed in ndc1. The PCA score plot indicated similar prenylquinone-lipid compositions for a representative 35S:NDC1-YFP line and the WT under moderate light conditions. This finding together with the absence of a visible phenotype suggests that the insertion of the 35S:NDC1-YFP encoding T-DNA construct was without positional effects.
To investigate the implication of VTE1 and NDC1 plants in prenylquinone metabolism under HL stress, we compared the lipid profiles of wild type plants with mutants after 4 and 8 days of HL exposure (Figure 4). Whereas vte1 was blocked at the DMPBQ stage, WT plants strongly accumulated antioxidant lipids: γ-, α-tocopherol, PQ, PQH2, and PC8. (Figures 4A,B). ndc1 differed from WT by the presence of the phylloquinone precursor and PQ but interestingly also by the accumulation of α-TQ (Figures 4C,D).

α-TOCOPHEROL QUINONE, AN INTERMEDIATE OF THE TOCOPHEROL REDOX CYCLE ACCUMULATES UNDER HL STRESS IN ndc1
To assess the quantitative impact of NDC1 and VTE1 on prenylquinones after HL stress, we quantified, using pure standards, the principal compounds that were distinguished by PCA: α-tocopherol and α-TQ. As expected under HL conditions, the levels of α-tocopherol ( Figure 5A) and oxidized α-TQ ( Figure 5B) increased in WT, about 3 (Student's t-test p D0-D8 = 0.0014) to 6-fold (p D0-D8 = 0.009) respectively after 8 days.
With regard to the concentration of oxidized α-TQ, pronounced differences were observed between WT and mutant plants. In WT, the concentration of α-TQ increased under HL stress. However, ndc1 accumulated at least 3 times as much α-TQ as WT under moderate and HL conditions. The finding for α-TQ was confirmed for two ndc1 T-DNA insertion alleles (Supplementary  Figure 1).
In contrast, in 35S:NDC1-YFP and 35S:VTE1-YFP the level of α-TQ remained unchanged after HL stress and was about 8 times lower than in the WT at D8.

NDC1 IS IMPLICATED IN THE REGENERATION OF REDUCED PQ AND THE FORMATION OF PC8
The concentrations of total PQ including the proportion of the oxidized and reduced forms ( Figure 6A) and of PC8 (Figures 6B,C) were measured.
In WT plants, the concentration of total PQ was unchanged after 8 days of HL (p D0-D8 = 0.471) while a slight decrease of PC8 was observed (p D0-D8 = 0.013).
In 35S:VTE1-YFP, the concentration of total PQ was generally lower than in the WT and this difference increased under HL (p D0 = 0.068, p D4 = 0.03, p D8 = 0.012). Concomitantly, PC8 concentration in 35S:VTE1-YFP was 10 times higher than WT under moderate light conditions and 27 times higher after 8 days of HL.
As expected, PC8 was not at all detectable in vte1 (data not shown).
ndc1 and 35S:NDC1-YFP, accumulated the highest concentrations of total PQ under HL. In ndc1, the concentration of oxidized PQ was significantly higher than in the WT after 8 days of HL (PQox p D8 ndc1-WT = 0.0078). In contrast, in 35S:NDC1-YFP, the accumulation of the reduced form PQH2 made the difference (PQred p D8 35S:NDC1-YFP − WT = 8.50e −5 ). As expected, the concentration of PC8 detected in ndc1 mutant plants was significantly lower than in the WT, but no difference was observed between WT and 35S:NDC1-YFP (p D8 WT-35S:NDC1-YFP = 0.1).

PLASTOGLOBULES, A MAJOR COMPARTMENT OF PRENYLQUINONE METABOLISM AND REPAIR
To analyze the distribution of the prenylquinones, we isolated chloroplasts from leafs of WT and ndc1 after 7 days of HL and separated the thylakoid membranes and the plastoglobules. We then carried out prenyllipid profiling on whole leafs, isolated chloroplast, thylakoids and plastoglobules (Figures 7A,B). The galactolipids, MG 1; monogalactosyldiglyceride (18:3/16:3) and MG 2; monogalactosyldiglyceride (18:3/18:3), abundant chloroplast membrane lipids contributing to the thylakoids as well as the lipid monolayer of plastoglobules, were used as an internal reference to assess the enrichment of prenylquinones. Clearly, the prenylquinone compounds were enriched in plastoglobules, i.e., small peaks for MG1 and −2, large peaks for α-tocopherol and PQ/PQH 2 when compared to the thylakoid membranes in both WT and ndc1 (approximately 30-and 40-fold, respectively). Using γ-tocopherol as an internal reference (Figure 7C), increase of α-TQ and decrease of PC8 were observed in ndc1 PG. For α-TQ, the enrichment in PG compared to thylakoid membranes was 3-and 5-fold, respectively in WT and ndc1. Note that α-TQ cannot be seen as peak in the chromatograms (Figures 7A,B) due to its relatively low abundance. As expected phylloquinone (Vit K) was detectable only in WT but not in ndc1 PG.

DISCUSSION
In this study we analyzed the dynamics of prenyl lipid metabolites during the change from moderate light to HL conditions using a non-targeted lipidomics approach. It is known that during acclimation to HL conditions, several prenylquinones accumulate in Arabidopsis leaves Szymanska and Kruk, 2010b;Eugeni Piller et al., 2011). The four Arabidopsis genotypes used in these experiments resulted in distinct prenylquinone signatures. The most typical compounds that accumulated in each of the respective lines were: DMPBQ in vte1, 2-phytyl-1,4-naphthoquinone in ndc1, δ-, γ-tocopherol and PC8 in 35S:VTE1-YFP and finally PQH 2 in 35S:NDC1-YFP overexpressing plants (Figure 3). Overall, α-tocopherol was the prenylquinone that increased most during the course of light stress among the 500 compounds analyzed, except in the vte1 mutant that lacks the tocopherol cyclase (Figure 4). This is testimony to the importance of α-tocopherol as a lipid antioxidant at the thylakoid membrane, which is subject to photooxidation and photoinhibition at PSII due to increased ROS production under HL stress . We previously showed that the α-tocopherol accumulation coincides with an increase in size and number of PG under HL stress (Martinis et al., 2014). This work demonstrates the implication of NDC1 in the tocopherol redox cycle. During HL stress, ndc1 mutant plants accumulate the α-tocopherol oxidation product, α-TQ ( Figure 4D). It has already been demonstrated that NDC1 is an enzyme with a wide specificity and able to reduce a range of quinolic substrates in vitro such as decyl-PQ, decyl-ubiquinone as well as in purified plastoglobules due to their contents of prenylquinones . For α-tocopherol recycling to proceed efficiently it is likely that α-TQ must be present in the reduced form (α-TQH 2 ). However, our current methodology does not allow the detection of α-TQH 2 . Nevertheless it is highly probable that NDC1 functions in the reduction of α-TQ to α-TQH 2 to regenerate α-tocopherol. An analogous reaction mechanism has been demonstrated for the formation of γ-tocopherol, in which VTE1 closes the chromanol ring preferentially in the reduced form of DMPBQ (Grutter et al., 2006). NDC1 is also implicated in the reduction of PQ to PQH 2 as 35S:NDC1-YFP plants exhibited higher PQH 2 /PQ ratios ( Figure 6A). Thus, NDC1 may directly influence the redox state of the PQ reservoir. Most likely, this increase in PQH 2 concerns primarily the proportion of the plastoquinone pool present in plastoglobules that is not directly implicated in photosynthesis . The observed increase of total plastoquinone in 35S:NDC1-YFP plants ( Figure 6A) may be necessary to maintain sufficient oxidized PQ to allow electron transport to proceed efficiently at the thylakoid membranes.
35S:VTE1-YFP plants showed a decrease of total PQ after HL stress (Figure 6A). It is important to note that VTE1 catalyses the production of PC8 from PQH 2 (Zbierzak et al., 2010). Therefore, the decrease of total PQ is readily explained by the pronounced accumulation of PC8 in this genotype ( Figure 6B). PQH 2 may continuously be siphoned off as a substrate of VTE1 to form PC8 explaining the decrease of PQ and the increase of PC8 in 35S:VTE1-YFP. However, in agreement with Szymanska and Kruk (2010a), our results show that the production of PC8 is not influenced by HL in WT, ndc1 and 35S:NDC1-YFP plants but an increase is apparent in 35S:VTE1-YFP plants.
Potentially, this could be explained by an increased flux of PQH 2 to PG under HL in the presence of elevated concentrations of VTE1-YFP.
In conclusion, we identify NDC1 as a novel enzyme that participates in the α-tocopherol redox cycle probably by reducing α-TQ to α-TQH 2 . In this recycling pathway, VTE1 was already known to convert TMPBQH 2 to α-tocopherol. By hosting both NDC1 and VTE1, PG appear to play a role as metabolic repair site in the tocopherol redox cycle. This hypothesis is supported by the enrichment of α-TQ in the PG of ndc1 compared to the WT ( Figure 7C). Also, the overexpression of both NDC1-YFP and VTE1-YFP suppressed the increase of α-TQ observed in the wild type under HL ( Figure 5B). This may be explained by higher levels of NDC1 or VTE1 activity that may affect the reaction kinetics of the tocopherol redox cycle and accelerate the reduction of α-TQ.
Beyond its role in tocopherol recycling and plastoquinone reduction, ndc1 lacks phylloquinone and accumulates its demethyl precursor instead ( Figure 4D) . This indicates that NDC1 has "moonlighting" role in the final methylation step of phylloquinone biosynthesis that is catalyzed by AtMENG. In the future, it will be of great interest to investigate the mechanisms of NDC1 in more detail and to determine the role of this unusual enzyme in other species.  Plants grown under moderate light conditions (D0) were exposed to continuous HL (500 μE m-2 s-1) for 4 (D4) and 7 days (D7). Data are means of three experiments (n = 3).