Journal of Photochemistry and Photobiology B: Biology 130 (2014) 68–75

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The chloroplast protein LTO1/AtVKOR is involved in the xanthophyll cycle and the acceleration of D1 protein degradation Zhi-Bo Yu a, Ying Lu a, Jia-Jia Du a, Jun-Jie Peng a, Xiao-Yun Wang a,b,⇑ a b

College of Life Science, Shandong Agricultural University, Shandong, Taian 271018, People’s Republic of China State Key Laboratory of Crop Biology, Shandong Agricultural University, Shandong, Taian 271018, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 27 July 2013 Received in revised form 11 October 2013 Accepted 5 November 2013 Available online 14 November 2013 Keywords: LTO1 Disulfide bond Photoinhibition Xanthophyll cycle D1 protein turnover Arabidopsis

a b s t r a c t The thylakoid protein LTO1/AtVKOR-DsbA is recently found to be an oxidoreductase involved in disulfide bond formation and the assembly of photosystem II (PSII) in Arabidopsis thaliana. In this study, experimental evidence showed that LTO1 deficiency caused severe photoinhibition which was related to the xanthophyll cycle and D1 protein degradation. The lto1-2 mutant was more sensitive to intense irradiance than wild type. When treated with different concentrations of dithiothreitol (DTT), an inhibitor of violaxanthin de-epoxidase (VDE) in the xanthophyll cycle, there was a larger reduction in NPQ in the wild type than in the lto1-2 mutant under high irradiance, indicating that lto1-2 had a lower sensitivity to DTT gradients than did the wild type. Zeaxanthin in the xanthophyll cycle, which participates in the thermal dissipation of excess absorbed light energy, was much less active in lto1-2 than in the wild type under intense light levels, and the de-epoxidation state of the xanthophyll cycle was consistent with the susceptibility of NPQ. Together these observations indicated that aggravated photoinhibition in lto1-2 was related to a reduction in xanthophyll cycle-associated energy dissipation. When D1 protein synthesis was suppressed by an inhibitor of chloroplast protein synthesis (streptomycin sulfate), the levels of D1 protein decreased more in the lto1-2 mutant than in the wild type when exposed to intense light levels, implying that a deficiency in LTO1 accelerated the degradation of D1 and thus affected D1 turnover. Transgenic complementation of plants with lto1-2 ultimately allowed for the recovery of the photoinhibition properties of leaves. Ó 2013 Elsevier B.V. All rights reserved.

1. Introduction Light energy is essential for photosynthesis and is the driving force for the synthesis of chemical energy. Light energy can also be harmful when absorption by chlorophylls exceeds the capacity for energy utilization in photosynthesis [1,2]. Some excess excitation energy is dissipated as fluorescence or heat, and some is transferred to molecular oxygen as the terminal electron acceptor in the noncyclic electron transport pathway instead of NADP+. This pathway generates highly damaging reactive oxygen species (ROS) that can lead to photoinhibition [3–5]. To avoid photoinhibition, photosynthetic organisms have developed various photoprotective mechanisms to resist photooxidative damage and to repair damaged protein components [6,7]. Excess absorbed energy is dissipated as heat to minimize excitation pressure on PSII. This process is called ‘High energy’ quenching (qE). The rapid formation of qE is a component of ⇑ Corresponding author. Address: College of Life Science, Shandong Agricultural University, Shandong, Taian 271000, People’s Republic of China. Tel.: +86 538 8242656x8430; fax: +86 538 8242217. E-mail address: [email protected] (X.-Y. Wang). 1011-1344/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jphotobiol.2013.11.003

non-photochemical quenching (NPQ) and is considered to be one of the most important photoprotective mechanisms [8]. qE primarily depends on the xanthophyll zeaxanthin (Zea), the accumulation of which requires the activity of violaxanthin de-epoxidase (VDE) in the xanthophyll cycle. Under excess light, epoxide xanthophyll violaxanthin (Vio) is rapidly converted via the intermediate antheraxanthin (Ant) to the de-epoxide zeaxanthin (Zea) under the action of VDE. In the dark, a reverse reaction occurs that converts Zea to Vio. Zea can directly quench the triplet-excited state of chlorophyll (3Chl) or it can favor proton-induced aggregation of the photosystem II light harvesting complex (LHCII) leading to excess energy dissipation [9]. Thus, Zea is effective at dissipating excess photon-energy and protecting plants from photoinhibition. The role of Zea-dependent quenching in NPQ in higher plants has been deduced from the linear relationship between Zea formation and the magnitude of NPQ. Zea synthesis-deficient mutants exhibit lower NPQ levels than do wild type plants [10]. As the key enzyme in the xanthophyll cycle, VDE is localized to the thylakoid lumen, activated by low pH, and has been shown to use ascorbate as its reductant in vitro [11]. The N-terminal region of VDE is cysteine-rich and contains 11 of the 13 cysteine residues present in the protein. Deletion of this cysteine rich N-terminal

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region result in total loss of activity [12]. The activity of VDE is compromised by a single base-pair mutation of Cysteine 72 to Tyrosine 72 in the npq1 mutant from Arabidopsis [13]. In addition, experiments have demonstrated that inhibition of VDE by DTT results in a great loss of PSII activity [14]. Recent research has found that VDE is captured as a thioredoxin target of the thylakoid lumen and that the activity of VDE is inhibited after the disulfide bond is reduced by thioredoxin [15]. However, the oxidative regulation of the enzyme has not been elucidated. The repair of PSII, which is a primary target of photooxidative damage in the photosynthetic apparatus, appears to be another photoprotective process [16]. As the core component of PSII, the Dl protein is easily damaged by high light levels [17,18]. The repair of damaged D1 protein in PSII primarily involves a cycle of degradation and re-synthesis [19–21]. Photoinhibition is related to the balance between the rate of photodamage to D1 and the rate of repair. When re-synthesis of D1 cannot keep pace with D1 protein degradation, net loss of PSII activity is observed [22]. Therefore, fast D1 protein turnover is also connected to photoinhibition in vivo [23–25]. Lumen thiol oxidoreductase1 (LTO1) in Arabidopsis thaliana is encoded by At4g35760 and is a homolog of vitamin K epoxide reductase (VKOR) in mammals, so it is also called AtVKOR. The first 45 amino acids from the N-terminus are found to act as a transit peptide that targets the protein to the chloroplast thylakoid. When this transit peptide is cut, the protein can catalyze the formation of disulfide bonds in E. coli [26]. LTO1 is a fusion protein containing two domains: an integral membrane domain homologous to the catalytic subunit of mammalian VKOR and a soluble DsbA-like/ Trx-like domain (AtDsbA) facing the oxidative thylakoid lumen [26]. LTO1 catalyzes the in vitro formation of disulfide bonds in the PsbO protein, a luminal subunit of the oxygen-evolving complex (OEC) in PSII [27]. Studies have also found that the redox activity of LTO1 is required for the assembly of PSII in Arabidopsis [27]. LTO1 deficiency causes a reduction in the amount of PSII subunits, including D1 and PsbO [27]. Whether LTO1 regulated the VDE-mediated xanthophyll cycle and D1 protein turnover in the photoprotective pathway was not clear. In the present study, we investigated the relationship between LTO1 and the two photoprotective pathways using the wild type, an LTO1 knock-out mutant line (lto1-2) and complementation plants. We found that the lto1-2 mutant suffered from severe photoinhibition under intense light conditions. LTO1 protein regulated xanthophyll cycle-mediated excess energy dissipation. The deficiency of LTO1 protein also accelerated the degradation rate of D1 and affected D1 protein turnover.

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previously described [29]. To ensure synchronized germination, the seeds were stratified for 48 h at 4 °C in the dark. 2.2. Inhibitor and high-light treatments For inhibitor uptake through the transpiration stream, leaves were detached and the petioles were soaked. Leaves of wild type, lto1-2 mutant and complementation plants had been dark-adapted for 24 h before absorption of inhibitor. For inhibition of violaxanthin de-epoxidation, predarkened leaves were fed with 3 mM, 5 mM and 10 mM dithiothreitol (DTT) and incubated in low light (30 lmol m2 s1) for 3 h at 22 °C. To prevent Dl protein synthesis, 1 mM streptomycin sulfate (SM) was fed to the leaves, which were then incubated in low light (30 lmol m2 s1) at 22 °C for 3 h. Control leaves were placed in water and kept in low light for approximately 3 h. For photoinhibition of leaves, detached control and inhibitor-fed leaves were illuminated at a PFD of 1000 lmol m2 s1 for 3 h. Actinic light measuring 1000 lmol m2 s1 was produced by the Chlorolab-2 light source at 25 °C, and the petioles were kept in the same solution as for the high-light treatments for 3 h. 2.3. Measurement of the chlorophyll fluorescence parameters Fluorescence induction of leaves was measured using a pulsemodulated fluorometer (FMS-2, Hansatech, UK) as previously described [29]. To achieve the maximum quantum yield of photosystem II (Fv/Fm) measurements, the leaves were darkened for 15 min following photoinhibition treatment. The following parameters were then calculated: Fv/Fm = (Fm  Fo)/Fm; UPSII = (Fm0  Fs)/Fm0 ; and NPQ = (Fm  Fm0 )/Fm0 . Every experiment at least six leaves were measured, and three independent experiments were accomplished. 2.4. Pigment extraction

2. Materials and methods

Pigments were extracted from wild type, lto1-2 mutant and complementation leaves. Intact leaves had been dark-adapted for 24 h. Then predarkened leaves (approximately 1 g fresh weight, three replicates) were fed with 0 mM, 3 mM and 5 mM DTT and incubated in low light (30 lmol m2 s1) for 3 h at 22 °C. Leaves were frozen in liquid nitrogen and stored at 70 °C. For extraction, the leaves were first ground in a mortar in liquid nitrogen. The resulting powder (0.5 g) was then added to 100% acetone (5 ml), vortexed, and centrifuged at 2500g for 10 min at 4 °C. The acetone supernatants were removed and filtered through a 0.45 lm syringe filter into centrifuge tubes. The extract was kept in the dark at 20 °C until analyzed.

2.1. Plant materials and growth conditions

2.5. HPLC determinations

The Col-0 ecotype of Arabidopsis served as the wild type. The A. thaliana lto1 T-DNA insertion mutant line CS858849 (lto1-2) was obtained from the Arabidopsis Biological Resource Center. Homozygous lto1-2 lines were screened and complementation plants were obtained according to the previous description [28]. Arabidopsis was grown in vermiculite under 120 lmol m2 s1 photon flux density (PFD) with short-day conditions (8 h of illumination and a 16 h dark cycle) at a constant temperature of 22 °C. 8-week-old wild type, lto1-2 mutant and complementation plants were used for the experiments. For growth on agar plates, seeds were surface-sterilized with 70% ethanol and 2.6% bleach for 5 min and 10 min, respectively. Then seeds were washed five times with sterilized water containing detergent Tween-20. The washed seeds were plated on MS medium containing 3% sucrose as

Pigment separation was performed in a high-performance liquid chromatography (HPLC) system (Waters, USA). Chromatography was carried out on a (250  U 4.6 mm, 5 lm) Waters Spherisorb C18, and the mobile phases were pumped by a Waters M45 high pressure pump at a flow rate of 1.4 ml/min. The column was equilibrated prior to injecting each sample by flushing with acetonitrile:methanol:Tris–HCl (0.05 mol/L, pH 7.5) (72:8:3, v/v/v, mobile phase A) for 30 min. Five-microliter samples were injected into the column, and the absorbance at 440 nm recorded. Mobile phase A was pumped for 4 min, linear solvent strength gradient was pumped for 2.5 min, and a mixture of methanol:hexane (5:1, v/v, mobile phase B) was then pumped for 16.5 min. Finally, mobile phase A was pumped for 5 min. Peaks were identified by standard methods described previously [30]. The de-epoxidation of the

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xanthophyll cycle was calculated (Ant + 2Zea)/(Vio + Ant + Zea).

as

the

peak

area:

2.6. Thylakoid membrane protein extraction and western blot analysis Thylakoid membranes were prepared as previously described by [31]. The chlorophyll content was determined according to previously described methods [32]. Sodium dodecyl sulfate–Polyacrylamide gel electrophoresis (SDS–PAGE) and western blot analysis were carried out as described previously [33]. Protein samples corresponding to equal amounts of chlorophyll were separated through 15% SDS–PAGE gels. Then, proteins were transferred onto Immobilon-P membranes (Millipore) and blotted with anti-D1 antibody. The D1 protein antibody was purchased from Genscript. A horseradish peroxidase-conjugated anti-rabbit antibody (CW0103) was used as the secondary antibody. The immune-decorated bands were detected by sensitive fluorography with enhanced chemiluminescence (Amersham, Japan). Signals of immunoblots were quantified using the ImageJ program. 3. Results 3.1. The deficiency of LTO1 protein enhanced photoinhibition during high irradiance stress Previous research has shown that LTO1 protein deficiency impacts the photosynthetic activity of PSII by limiting electron flow, and the knock-down lto1-1 mutant seems to suffer from higher levels of photoinhibition than the wild type under light levels suitable for growth (170 lE m2 s1) [27]. Here, to confirm whether the loss of LTO1 caused severe photoinhibition under high light conditions, we compared the chlorophyll fluorescence parameters of knock-out mutant lto1-2 lines with wild type and complementation plants using a pulse-modulated fluorometer (FMS-2). Under growth light conditions (before high light treatment), the maximum efficiency of the PSII photochemistry (Fv/Fm) and the effective quantum yield of PSII (UPSII) in the lto1-2 mutant line were lower than those of wild type. When the leaves were exposed to an irradiance of 1000 lmol m2 s1 for 1 h, these parameters decreased sharply both in the lto1-2 mutant and the wild type. As the time prolonged to 2 h, the values decreased continuously (Fig. 1a and b). The Fv/Fm ratio fell from 0.53 to 0.30, a 43% reduction, in the lto1-2 mutant and from 0.84 to 0.70, a 17% reduction, in the wild type with 1 h of irradiance (Fig. 1a). Changes in the UPSII ratio followed a similar trend to the Fv/Fm ratio (Fig. 1b). As the high irradiance time was extended to 2 h, the decrease in photosynthetic activity was enhanced. The lto1-2 mutant was more sensitive to strong light than the wild type, as was indicated by the more pronounced decrease in Fv/Fm and UPSII. This result suggested that the lto1-2 mutant suffered from severe photoinhibition. To investigate whether photoinhibition in the lto1-2 mutant was related to the energy dissipated through the xanthophyll cycle under strong light conditions, a potent and widely used inhibitor of VDE, DTT, was used [34,14]. Compared to the control treatment (no DTT), the reductions in Fv/Fm and UPSII were enhanced under high irradiance with the DTT treatment both in the lto1-2 mutant and the wild type. The values of Fv/Fm and UPSII decreased as DTT concentrations increased (Fig. 1a and b). The Fv/Fm ratio in the lto1-2 mutant decreased more significantly than the wild type after DTT treatment, suggesting that the lto1-2 mutant suffered from severe photoinhibition. The reduction in UPSII level in the wild type was more pronounced than for the lto1-2 mutant during exposure to high irradiance, especially after 1 h of treatment, indicating that the lto1-2 mutant was less sensitive to DTT than the wild type. Transgenic complementation ultimately recuperated the charac-

Fig. 1. Chlorophyll fluorescence parameters. Mean values ± the standard deviation of three independent experiments were reported. Chlorophyll fluorescence parameters were measured in 8-week-old leaves. After 24 h in the dark, leaves were untreated (d,s) or treated with 3 mM DTT (.,4), 5 mM DTT (j,h) and 10 mM DTT (,}) at low light (30 lmol m2 s1) for 3 h at 22 °C. Then, leaves were exposed to an irradiance of 1000 lmol m2 s1. (a) The initial value of the wild type leaves before treatment was taken as 100%. The results were reported as percentages. (b) The relative value of UPSII. Solid lines and solid symbols represent the WT, and dashed lines and hollow symbols represent the lto1-2 mutant.

teristic changes described above (Tables S1–S2). Based on the results above, it seems reasonable to assume that the aggravated photoinhibition observed in the lto1-2 mutant may be related to the xanthophyll cycle. However, whether the suppression of xanthophyll cycle was caused by inhibition of VDE needed to be investigated. 3.2. The inhibitor of VDE in the xanthophyll cycle altered non-photochemical quenching (NPQ) The xanthophyll cycle is an efficient thermal dissipation mechanism in plants exposed to high light. The change in the amount of the de-epoxidated xanthophyll zeaxanthin is related to the extent of heat dissipation measured as NPQ. Under growth light, the NPQ values were lower in the lto1-2 mutant (0.86 ± 0.03) than in wild-type plants (1.2 ± 0.04) (Fig. 2a). After high irradiance for 1 h, the NPQ values increased to 1.4 and 1.72 in the lto1-2 mutant and the wild type, respectively. The low NPQ value in the lto1-2 mutant suggested a low capability for the dissipation of excess absorbed light energy. The effects of DTT on NPQ under high irradiance were further investigated. Compared to the untreated control, the addition of 3 mM DTT caused a significant decrease in NPQ in the wild type and in the lto1-2 mutant after high irradiance for 1 h, as indicated by the fact that the NPQ values fell from 1.72 to 0.96 in the wildtype and from 1.4 to 1.0 in the lto1-2 mutant (Fig. 2a). Increasing

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DEI values were lower in the lto1-2 mutant line than in the wild type and complementation plants. This result suggested that deepoxidation of Vio to Zea via Ant was suppressed in the lto1-2 mutant. Although DTT is nonspecific inhibitor of VDE, the significant DTT-induced reduction in DEI observed in the lto1-2 mutant and wild type suggested that VDE was inhibited by DTT. Three millimolar DTT almost completely inhibited the formation of Zea. The DEI values were little difference among in the lto1-2 mutant, wild type and complementation plants. Changes in the DEI were consistent with changes in NPQ. The results above indicated that the LTO1 protein was involved in regulation of the xanthophyll cycle, and then regulating Zea-dependent energy dissipation in the photoprotective mechanism. 3.4. The maximum efficiency of PSII photochemistry was affected when treated with an inhibitor of protein synthesis in chloroplast

Fig. 2. Changes in non-photochemical fluorescence quenching (NPQ) and the deepoxidation state of xanthophyll cycle pigments. After 24 h in the dark, 1eaves were fed with 0 mM, 3 mM and 5 mM DTT at low light (30 lmol m2 s1) for 3 h at 22 °C. Leaves were then exposed to an irradiance of 1000 lmol m2 s1 for 1 h. (a) The change of non-photochemical quenching for the WT, lto1-2 and lto1-2C plants. lto12C represents transgenic complementation. 0 h indicates that the leaves were placed in growth light for 1 h as a control. (b) The change of the de-epoxidation state. 0 h of light indicate that leaves were placed in the dark for 24 h as a control. The results were reported as the mean of three independent experiments.

the DTT concentration enhanced the reduction in NPQ values. A similar trend in NPQ values was observed both in transgenic complementation and wild type plants. Because the extent of the reduction in NPQ in the lto1-2 mutant was less than in the wild type, the lto1-2 mutant had lower sensitivity to DTT than the wild type. Then, the NPQ values for the lto1-2 mutant were slightly higher than the wild type after treatment with different concentrations of DTT. 3.3. The LTO1 protein was related to the de-epoxidation of the xanthophyll cycle The great reduction in NPQ induced by DTT may be associated with a reduction in the de-epoxidation of the pigment interconversion within the xanthophyll cycle. We identified changes in the concentrations of violaxanthin (Vio), antheraxanthin (Ant), and zeaxanthin (Zea) using high-performance liquid chromatography (HPLC). The de-epoxidation state of the xanthophyll cycle pigments was described using the de-epoxidation index (DEI) [30]. After dark treatment for 24 h, the DEI values were very low (approximately 0.07) (Fig. 2b) because Zea has been converted to Vio. As the key enzyme of xanthophyll cycle, VDE can only be activated by light and the light-driven transmembrane proton gradient to promote conversion of Vio to Zea via Ant. When leaves were exposed to 1000 lmol m2 s1 irradiance for 1 h, the DEI values increased both in the lto1-2 mutant and in the wild type, but the

The previous investigation has shown that the redox activity of LTO1 is indispensable for the assembly of PSII [27]. The D1 protein level in PSII is significantly reduced in the lto1-2 mutant [28]. The results in Fig. 2a also showed that the NPQ values of the lto1-2 mutant were slightly higher than the wild type after treatment with different concentrations of DTT under high light conditions. These data suggested that there were other pathways of energy dissipation in addition to the xanthophyll cycle being impacted by the LTO1 deficiency. The photoprotective pathway of D1 turnover may be affected in the lto1-2 mutant. Previous work has shown that the synthesis of D1 is not affected in the lto1-2 mutant [28]; rather, the decrease in D1 protein concentration may be due to enhanced degradation. To verify this hypothesis, SM, an inhibitor of protein synthesis in chloroplast, was used. As D1 protein is chloroplast-encoded protein, so its synthesis is effectively prevented by SM [35]. Under growth light, the Fv/Fm ratio decreased in both the lto1-2 mutant and the wild type in the presence of SM (Fig. 3a). However, the decline in Fv/Fm in the lto1-2 mutant was more than the decline in the wild type, and the difference in Fv/Fm between the lto1-2 mutant and wild type increased as the irradiation time increased (Fig. 3b). This result indicated that the lto1-2 mutant had a photodamage to PSII. Fig. 3c showed the changes in the ratio of Fv/Fm following exposure of leaves to strong light and treatment with SM. Compared with changes in the Fv/Fm ratio without SM treatment, a more significant decrease in the Fv/Fm ratio was observed both in the lto1-2 mutant and wild type. After high irradiance treatment for 3 h following SM treatment, the Fv/Fm ratio decreased to 0.02 in the lto1-2 mutant compared to 0.19 in the wild type. To compare the decrease of Fv/Fm in wild type with that in lto1-2 mutant easily, the relative Fv/Fm ratio (the Fv/Fm ratio of SM-treated leaves divided by the Fv/Fm ratio of control leaves) was compared by defining the initial values of Fv/Fm (before high irradiance) as 100% in the lto1-2 mutant and wild type. The percentage of Fv/Fm was significantly lower in the lto1-2 mutant than that in the wild type (Fig. 3d), indicating that the lto1-2 mutant had a severe photodamage to PSII. Transgenic complementation ultimately demonstrated that a photodamage to PSII in the lto1-2 mutant plants was due to LTO1 (Tables S3–S4). These results suggested that LTO1 was involved in another photoprotective pathway, D1 turnover. 3.5. The deficiency of LTO1 protein accelerated the D1 protein degradation rate To further investigate whether a severe photodamage to PSII in lto1-2 mutant was related to the photoprotective pathway of D1 turnover, the quantity of D1 protein in the lto1-2 mutant, the wild

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Fig. 3. A photodamage to PSII (estimated from a reduction in Fv/Fm) was measured. The results were reported as the mean of three independent experiments. The leaves were treated either in the absence (d,.) or presence (s,4) of 1 mM SM at low light for 3 h at 22 °C and were then exposed to either an irradiance of 120 lmol m2 s1 (growth light, a and b) or an irradiance of 1000 lmol m2 s1 (high light, c and d) for 3 h. Fv/Fm was measured after 15 min of dark adaption at room temperature. a and c: The absolute value of Fv/Fm. b and d: The relative value from a and c. The initial value before illumination was taken as 100%, and the results were reported as percentages. Solid and dashed lines represent the wild type and the lto1-2 mutant, respectively.

type and complementation plants was analyzed by western blot. Recently research has shown that in lto1-2 mutants, D1 protein has only about half as much as in wild-type by using concentration gradient of the sample proteins under growth light [28]. Our results were consistent with previous investigation (Fig. 4a and b). Treatments with SM in low light (LL) for 3 h did not significantly alter the D1 level in the lto1-2 mutant, wild type or complementation plants (Fig. 4a and b). After SM treatment, the leaves were exposed to a high light (HL) level of 1000 lmol m2 s1 for 3 h. D1 protein in the PSII centers induced by high light levels was rapidly degraded. There were only trace amounts of D1 protein in the lto1-2 mutant and yet relatively high quantities both in the wild type and compensation plants (Fig. 4a and b), indicating that there was more degradation of D1 protein in the lto1-2 mutant than in the wild type. After treatment with SM and exposure to growth light (GL) for 3 h, Dl protein was also degraded both in the lto1-2 mutant and the wild type, but much less than that the degradation under high light with SM treatment. However, the degradation of Dl protein in the lto1-2 mutant was much more than that in the wild type and compensation plants under this condition (Fig. 4a and b). Transgenic complementation plants had even more D1 protein than the wild type, showing that D1 protein was overexpressed in complementation plants. Changes in the level of D1 under different illumination intensities were consistent with changes in the Fv/Fm parameter,

suggesting that LTO1 protein deficiency accelerated the degradation of D1 protein and affected the photoprotective pathway of D1 turnover. 4. Discussion 4.1. Role of LTO1 in the regulation of energy dissipation depending on the xanthophyll cycle High light stress often inhibits photosynthesis and causes a reduction in Fv/Fm and UPSII during the daytime in the summer season [36]. In this investigation, the mutant lto1-2 line was shown to be more sensitive to high light (Fig. 1a and b). This result was consistent with previous research which has found that the lto1-1 mutant has more severe photoinhibition [27]. In general, sensitivity to photoinhibition is thought to be governed by various factors including the ability to synthesize chemical energy, the efficiency of various mechanisms that dissipate excess excitation energy to a harmless form and the capacity to repair photoinhibited PSII during illumination [37]. A previous research has suggested that NPQ is higher in the lto1-1 mutant (1.82 ± 1.15) than that in wild type (1.37 ± 0.06). But the value of NPQ in the lto1-1 leaves exhibits enormous variation and it is hardly to get a solid conclusion. ‘‘It was not consistently higher or lower than wild-type leaves’’ as they mention in

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mutant had less UPSII than did the wild type under different illumination intensities (Fig. 1b), indicating that the ETR within PSII was slower in the lto1-2 mutant. A reduction in the ETR might further inhibit the development of the transmembrane pH gradient [42], while VDE is activated by the reduction in the pH in the lumen during photosynthetic electron transport [43,44]. Thus, it was possible that the activity of VDE might also be affected due to the changes in the pH in the thylakoid lumen in the lto1-2 mutant. It seems that cyclic electron flow around photosystem I (PSI) may, to some extent, generate a transmembrane pH gradient when PSII electron transport is limited [45]. To summarize our results, the decrease of de-epoxidation state of the xanthophyll cycle pigments in lto1-2 mutant was related to the decrease of VDE activity, while the deletion of LTO1 in the mutant may affect either the redox state of VDE or a change in the proton gradient across thylakoid membranes or both. This issue requires further investigation.

4.2. LTO1 protein was involved in the process of Dl protein turnover Fig. 4. Immunoblot analysis of D1 degradation under growth light or in high light. Thylakoid membrane proteins (1 lg chlorophyll) were separated using SDS–PAGE, and the blot was probed with an anti-D1 polyclonal antibody. SM: streptomycin sulfate. a: GL: growth light with no SM. LL: low light for 3 h with 1 mM SM. GL: low light for 3 h with 1 mM SM and then growth light for 3 h. HL: low light for 3 h with 1 mM SM and then high light for 3 h. b: Signals of immunoblots were quantified using the ImageJ program. To compare D1 levels, ratios of WT were adjusted to 1 under growth light and without SM treatment. The relative levels of D1 compared to WT were calculated. The results were reported from three independent experiments. I: growth light with no SM. II: low light for 3 h with 1 mM SM. III: low light for 3 h with 1 mM SM and then growth light for 3 h. IV: low light for 3 h with 1 mM SM and then high light for 3 h.

the article [27]. While our results obtained in the present study have demonstrated that the lto1-2 mutant had lower NPQ values than that of wild type under growth light (120 lmol m2 s1) and high light (1000 lmol m2 s1) conditions for 1 h after 24 h darkness by extensive experiments (Fig. 2a), suggesting that the ability to dissipate excess absorbed light energy had been diminished for lto1-2 mutant plants. When leaves were fed with DTT, the inhibitor of VDE in the xanthophyll cycle, NPQ values decreased significantly in both the wild type and the lto1-2 mutant after high irradiance for 1 h. Previous research has also shown that the lack of Zea could conceivably affect a Zea-dependent sustained form of thermal energy dissipation when ascorbate is deficient and VDE is muted [38]. The lto1-2 mutant had lower sensitivity to DTT than the wild type under high irradiance. Therefore, the contribution of energy dissipation associated with the xanthophyll cycle was less in the lto1-2 mutant than in the wild type under this condition. HPLC analysis showed that the de-epoxidation state of pigments in the xanthophyll-cycle was also lower in the lto1-2 mutant than in wild type under high irradiance (Fig. 2b), suggesting that the lto1-2 mutant had reduced xanthophyll cycle activity compared with the wild type. A significant DTT-induced decrease in the DEI was observed both in the lto1-2 mutant and the wild type under high irradiance. Previous experiments have demonstrated that DTT inhibits the HL-stimulated formation of zeaxanthin [39]. Exogenous reduced glutathione (GSH) also markedly suppress the formation of Zea via the xanthophyll cycle [40]. Consequently, the activity of VDE, the key enzyme in the xanthophyll cycle, is dependent on disulfide bond, while LTO1 is recently found to play a role in promoting the disulfide bond formation in the thylakoid lumen [27]. We inferred that the disulfide bond-promoting function of LTO1 affected the activity of VDE and thus influenced the photoprotective mechanism of the xanthophyll cycle. According to the definition of transfer rates (ETR), ETR is determined by UPSII and light density [41]. From our results, the lto1-2

It is generally accepted that fast Dl turnover induced by high light occurs in higher plants. This phenomenon has been demonstrated with radioactive label incorporation into Dl or degradation of relabeled protein [35]. Dl protein degradation and the insertion of newly synthesized Dl protein into the reaction center complex is closely synchronized [46]. However, when the D1 protein degradation rate exceeds the rate of synthesis, photoinhibition of PSII occurs [47]. Previous studies have shown that the synthesis of D1 is not affected in the lto1-2 mutant both at the levels of transcription and translation in growth light. However, under this condition, the lto1-2 mutant has less D1 protein than wild type [28]. It was possible that its degradation rate was higher in the lto1-2 mutant than in wild-type plants. In this study, we used an inhibitor (SM) of protein synthesis in chloroplast, to focus on the degradation step of the repair cycle of the PSII reaction center. Our results showed that more intensive degradation of D1 protein took place in the lto1-2 mutant than in the wild type (Fig. 4a and b). LTO1 protein is involved in the stability of PSII and can promote the formation of disulfide bond in PsbO in vitro [27]. It is well known that the extrinsic PsbO requires intra-molecular disulfide bonds for its stabilization. Without the disulfide bonds probably in the absence of LTO1 in the lto1-2 mutant, the PsbO cannot bind to the PSII core and thus may destabilize the whole PSII complex. Then the degradation of core protein D1of PSII may become more easily to occur. Consequently, the lack of LTO1 may be responsible for accumulation of small amounts of the D1 protein (that means accelerated degradation of the D1 protein) under light stress. Furthermore, deficiency of PsbD or PsbE in Chlamydomonas reinhardtii leads to a reduction in the level of D1, though transcript levels of the D1 synthesis are not affected [48,49]. A similar phenomenon is also found in Arabidopsis for the HCF243 protein. The deficiency of this protein has no severe effects on the translation of D1 but appears to accelerate its degradation instead [50]. In summary, LTO1 protein deficiency affected the stability of PSII, the instability of which facilitated the degradation of the D1 protein. This deficiency eventually led to damage to the photoprotective pathway of D1 turnover. As to the relationship of xanthophyll cycle and D1 protein turnover, some studies have demonstrated that the inactivation of PSII reaction centers occurs as D1 turnover is inhibited, which depress the xanthophyll cycle by affecting transmembrane pH gradient [51,52]. While the impairment of xanthophyll cycle can accelerate photoinhibition in reverse by inhibiting the repair of photodamaged D1 protein [53]. The results above were consistent with our investigation. LTO1 protein was involved both photoprotective mechanisms in the xanthophyll cycle and Dl protein turnover.

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5. Abbreviations

Ant DEI DTT UPSII ETR GL qE HPLC HL LL LTO1 NPQ OEC PSII PSI LHCII FMS-2 PFD PAGE pH D1 ROS SM SDS Fv/Fm Vio VKOR VDE Zea

antheraxanthin de-epoxidation index dithiothreitol effective quantum yield of PSII electron transfer rate growth light ‘high-energy’ fluorescence quenching high-performance liquid chromatography high light low light Lumen Thiol Oxidoreductase1 non-photochemical quenching oxygen evolving complex photosystem II photosystem I photosystem II light harvesting complex pulse-modulated fluorometer photon flux density polyacrylamide gel electrophoresis potential of hydrogen reaction center protein of PSII reactive oxygen species streptomycin sulfate sodium dodecyl sulfate the maximum efficiency of PSII photochemistry violaxanthin vitamin K epoxide reductase violaxanthin de-epoxidase zeaxanthin

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AtVKOR is involved in the xanthophyll cycle and the acceleration of D1 protein degradation.

The thylakoid protein LTO1/AtVKOR-DsbA is recently found to be an oxidoreductase involved in disulfide bond formation and the assembly of photosystem ...
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