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

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Journal of Photochemistry and Photobiology B: Biology

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The chloroplast protein LTO1/AtVKOR is involved in the xanthophyllcycle and the acceleration of D1 protein degradation

1011-1344/$ - see front matter � 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.jphotobiol.2013.11.003

⇑ Corresponding author. Address: College of Life Science, Shandong AgriculturalUniversity, Shandong, Taian 271000, People’s Republic of China. Tel.: +86 5388242656x8430; fax: +86 538 8242217.

E-mail address: xyunwang@sdau.edu.cn (X.-Y. Wang).

Zhi-Bo Yu a, Ying Lu a, Jia-Jia Du a, Jun-Jie Peng a, Xiao-Yun Wang a,b,⇑a College of Life Science, Shandong Agricultural University, Shandong, Taian 271018, People’s Republic of Chinab 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 a b s t r a c t

Article history:Received 27 July 2013Received in revised form 11 October 2013Accepted 5 November 2013Available online 14 November 2013

Keywords:LTO1Disulfide bondPhotoinhibitionXanthophyll cycleD1 protein turnoverArabidopsis

The thylakoid protein LTO1/AtVKOR-DsbA is recently found to be an oxidoreductase involved in disulfidebond formation and the assembly of photosystem II (PSII) in Arabidopsis thaliana. In this study, experi-mental evidence showed that LTO1 deficiency caused severe photoinhibition which was related to thexanthophyll cycle and D1 protein degradation. The lto1-2 mutant was more sensitive to intense irradi-ance than wild type. When treated with different concentrations of dithiothreitol (DTT), an inhibitor ofviolaxanthin de-epoxidase (VDE) in the xanthophyll cycle, there was a larger reduction in NPQ in the wildtype than in the lto1-2 mutant under high irradiance, indicating that lto1-2 had a lower sensitivity to DTTgradients than did the wild type. Zeaxanthin in the xanthophyll cycle, which participates in the thermaldissipation of excess absorbed light energy, was much less active in lto1-2 than in the wild type underintense light levels, and the de-epoxidation state of the xanthophyll cycle was consistent with the suscep-tibility of NPQ. Together these observations indicated that aggravated photoinhibition in lto1-2 wasrelated to a reduction in xanthophyll cycle-associated energy dissipation. When D1 protein synthesiswas suppressed by an inhibitor of chloroplast protein synthesis (streptomycin sulfate), the levels of D1protein 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 photoinhi-bition properties of leaves.

� 2013 Elsevier B.V. All rights reserved.

1. Introduction

Light energy is essential for photosynthesis and is the drivingforce for the synthesis of chemical energy. Light energy can alsobe harmful when absorption by chlorophylls exceeds the capacityfor energy utilization in photosynthesis [1,2]. Some excess excita-tion energy is dissipated as fluorescence or heat, and some is trans-ferred to molecular oxygen as the terminal electron acceptor in thenoncyclic electron transport pathway instead of NADP+. This path-way generates highly damaging reactive oxygen species (ROS) thatcan lead to photoinhibition [3–5]. To avoid photoinhibition, photo-synthetic organisms have developed various photoprotectivemechanisms to resist photooxidative damage and to repair dam-aged protein components [6,7].

Excess absorbed energy is dissipated as heat to minimizeexcitation pressure on PSII. This process is called ‘High energy’quenching (qE). The rapid formation of qE is a component of

non-photochemical quenching (NPQ) and is considered to be oneof the most important photoprotective mechanisms [8]. qE primar-ily depends on the xanthophyll zeaxanthin (Zea), the accumulationof which requires the activity of violaxanthin de-epoxidase (VDE)in the xanthophyll cycle. Under excess light, epoxide xanthophyllviolaxanthin (Vio) is rapidly converted via the intermediateantheraxanthin (Ant) to the de-epoxide zeaxanthin (Zea) underthe action of VDE. In the dark, a reverse reaction occurs that con-verts Zea to Vio. Zea can directly quench the triplet-excited stateof chlorophyll (3Chl) or it can favor proton-induced aggregationof the photosystem II light harvesting complex (LHCII) leading toexcess energy dissipation [9]. Thus, Zea is effective at dissipatingexcess photon-energy and protecting plants from photoinhibition.The role of Zea-dependent quenching in NPQ in higher plants hasbeen deduced from the linear relationship between Zea formationand the magnitude of NPQ. Zea synthesis-deficient mutants exhibitlower NPQ levels than do wild type plants [10].

As the key enzyme in the xanthophyll cycle, VDE is localized tothe thylakoid lumen, activated by low pH, and has been shown touse ascorbate as its reductant in vitro [11]. The N-terminal regionof VDE is cysteine-rich and contains 11 of the 13 cysteine residuespresent in the protein. Deletion of this cysteine rich N-terminal

Z.-B. Yu et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 68–75 69

region result in total loss of activity [12]. The activity of VDE iscompromised by a single base-pair mutation of Cysteine 72 toTyrosine 72 in the npq1 mutant from Arabidopsis [13]. In addition,experiments have demonstrated that inhibition of VDE by DTT re-sults in a great loss of PSII activity [14]. Recent research has foundthat VDE is captured as a thioredoxin target of the thylakoid lumenand that the activity of VDE is inhibited after the disulfide bond isreduced by thioredoxin [15]. However, the oxidative regulation ofthe enzyme has not been elucidated.

The repair of PSII, which is a primary target of photooxidativedamage in the photosynthetic apparatus, appears to be anotherphotoprotective process [16]. As the core component of PSII, theDl protein is easily damaged by high light levels [17,18]. The repairof damaged D1 protein in PSII primarily involves a cycle of degra-dation and re-synthesis [19–21]. Photoinhibition is related to thebalance between the rate of photodamage to D1 and the rate of re-pair. When re-synthesis of D1 cannot keep pace with D1 proteindegradation, net loss of PSII activity is observed [22]. Therefore,fast D1 protein turnover is also connected to photoinhibitionin vivo [23–25].

Lumen thiol oxidoreductase1 (LTO1) in Arabidopsis thaliana isencoded by At4g35760 and is a homolog of vitamin K epoxidereductase (VKOR) in mammals, so it is also called AtVKOR. The first45 amino acids from the N-terminus are found to act as a transitpeptide that targets the protein to the chloroplast thylakoid. Whenthis transit peptide is cut, the protein can catalyze the formation ofdisulfide bonds in E. coli [26]. LTO1 is a fusion protein containingtwo domains: an integral membrane domain homologous to thecatalytic 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 inthe PsbO protein, a luminal subunit of the oxygen-evolving com-plex (OEC) in PSII [27]. Studies have also found that the redoxactivity of LTO1 is required for the assembly of PSII in Arabidopsis[27]. LTO1 deficiency causes a reduction in the amount of PSII sub-units, including D1 and PsbO [27]. Whether LTO1 regulated theVDE-mediated xanthophyll cycle and D1 protein turnover in thephotoprotective pathway was not clear.

In the present study, we investigated the relationship betweenLTO1 and the two photoprotective pathways using the wild type,an LTO1 knock-out mutant line (lto1-2) and complementationplants. We found that the lto1-2 mutant suffered from severe pho-toinhibition under intense light conditions. LTO1 protein regulatedxanthophyll cycle-mediated excess energy dissipation. The defi-ciency of LTO1 protein also accelerated the degradation rate ofD1 and affected D1 protein turnover.

2. Materials and methods

2.1. Plant materials and growth conditions

The Col-0 ecotype of Arabidopsis served as the wild type. The A.thaliana lto1 T-DNA insertion mutant line CS858849 (lto1-2) wasobtained from the Arabidopsis Biological Resource Center. Homo-zygous lto1-2 lines were screened and complementation plantswere obtained according to the previous description [28]. Arabid-opsis was grown in vermiculite under 120 lmol m�2 s�1 photonflux density (PFD) with short-day conditions (8 h of illuminationand a 16 h dark cycle) at a constant temperature of 22 �C.8-week-old wild type, lto1-2 mutant and complementation plantswere used for the experiments. For growth on agar plates, seedswere surface-sterilized with 70% ethanol and 2.6% bleach for5 min and 10 min, respectively. Then seeds were washed five timeswith sterilized water containing detergent Tween-20. The washedseeds were plated on MS medium containing 3% sucrose as

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, leaveswere detached and the petioles were soaked. Leaves of wild type,lto1-2 mutant and complementation plants had been dark-adaptedfor 24 h before absorption of inhibitor. For inhibition of violaxan-thin de-epoxidation, predarkened leaves were fed with 3 mM,5 mM and 10 mM dithiothreitol (DTT) and incubated in low light(30 lmol m�2 s�1) for 3 h at 22 �C. To prevent Dl protein synthesis,1 mM streptomycin sulfate (SM) was fed to the leaves, which werethen incubated in low light (30 lmol m�2 s�1) at 22 �C for 3 h. Con-trol leaves were placed in water and kept in low light for approx-imately 3 h. For photoinhibition of leaves, detached controland inhibitor-fed leaves were illuminated at a PFD of1000 lmol m�2 s�1 for 3 h. Actinic light measuring1000 lmol m�2 s�1 was produced by the Chlorolab-2 light sourceat 25 �C, and the petioles were kept in the same solution as forthe high-light treatments for 3 h.

2.3. Measurement of the chlorophyll fluorescence parameters

Fluorescence induction of leaves was measured using a pulse-modulated fluorometer (FMS-2, Hansatech, UK) as previouslydescribed [29]. To achieve the maximum quantum yield ofphotosystem II (Fv/Fm) measurements, the leaves were darkenedfor 15 min following photoinhibition treatment. The followingparameters were then calculated: Fv/Fm = (Fm � Fo)/Fm; UPSII =(Fm0 � Fs)/Fm0; and NPQ = (Fm � Fm0)/Fm0. Every experiment atleast six leaves were measured, and three independent experi-ments were accomplished.

2.4. Pigment extraction

Pigments were extracted from wild type, lto1-2 mutant andcomplementation leaves. Intact leaves had been dark-adapted for24 h. Then predarkened leaves (approximately 1 g fresh weight,three replicates) were fed with 0 mM, 3 mM and 5 mM DTT andincubated in low light (30 lmol m�2 s�1) for 3 h at 22 �C. Leaveswere frozen in liquid nitrogen and stored at �70 �C. For extraction,the leaves were first ground in a mortar in liquid nitrogen. Theresulting powder (0.5 g) was then added to 100% acetone (5 ml),vortexed, and centrifuged at 2500g for 10 min at 4 �C. The acetonesupernatants were removed and filtered through a 0.45 lm syringefilter into centrifuge tubes. The extract was kept in the dark at�20 �C until analyzed.

2.5. HPLC determinations

Pigment separation was performed in a high-performance liquidchromatography (HPLC) system (Waters, USA). Chromatographywas carried out on a (250 �U 4.6 mm, 5 lm) Waters SpherisorbC18, and the mobile phases were pumped by a Waters M45 highpressure pump at a flow rate of 1.4 ml/min. The column was equil-ibrated prior to injecting each sample by flushing with acetoni-trile:methanol:Tris–HCl (0.05 mol/L, pH 7.5) (72:8:3, v/v/v, mobilephase A) for 30 min. Five-microliter samples were injected intothe column, and the absorbance at 440 nm recorded. Mobile phaseA was pumped for 4 min, linear solvent strength gradient waspumped 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, mobilephase A was pumped for 5 min. Peaks were identified by standardmethods described previously [30]. The de-epoxidation of the

70 Z.-B. Yu et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 68–75

xanthophyll cycle was calculated as the peak area:(Ant + 2Zea)/(Vio + Ant + Zea).

2.6. Thylakoid membrane protein extraction and western blot analysis

Thylakoid membranes were prepared as previously describedby [31]. The chlorophyll content was determined according to pre-viously described methods [32]. Sodium dodecyl sulfate–Poly-acrylamide gel electrophoresis (SDS–PAGE) and western blotanalysis were carried out as described previously [33]. Proteinsamples corresponding to equal amounts of chlorophyll were sep-arated through 15% SDS–PAGE gels. Then, proteins were trans-ferred onto Immobilon-P membranes (Millipore) and blottedwith anti-D1 antibody. The D1 protein antibody was purchasedfrom Genscript. A horseradish peroxidase-conjugated anti-rabbitantibody (CW0103) was used as the secondary antibody. The im-mune-decorated bands were detected by sensitive fluorographywith enhanced chemiluminescence (Amersham, Japan). Signals ofimmunoblots were quantified using the ImageJ program.

Fig. 1. Chlorophyll fluorescence parameters. Mean values ± the standard deviationof three independent experiments were reported. Chlorophyll fluorescence param-eters were measured in 8-week-old leaves. After 24 h in the dark, leaves wereuntreated (d,s) or treated with 3 mM DTT (.,4), 5 mM DTT (j,h) and 10 mM DTT(�,}) at low light (30 lmol m�2 s�1) for 3 h at 22 �C. Then, leaves were exposed toan irradiance of 1000 lmol m�2 s�1. (a) The initial value of the wild type leavesbefore 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, anddashed lines and hollow symbols represent the lto1-2 mutant.

3. Results

3.1. The deficiency of LTO1 protein enhanced photoinhibition duringhigh irradiance stress

Previous research has shown that LTO1 protein deficiency im-pacts the photosynthetic activity of PSII by limiting electron flow,and the knock-down lto1-1 mutant seems to suffer from higherlevels of photoinhibition than the wild type under light levels suit-able for growth (170 lE m�2 s�1) [27]. Here, to confirm whetherthe loss of LTO1 caused severe photoinhibition under high lightconditions, we compared the chlorophyll fluorescence parametersof knock-out mutant lto1-2 lines with wild type and complementa-tion plants using a pulse-modulated fluorometer (FMS-2).

Under growth light conditions (before high light treatment), themaximum efficiency of the PSII photochemistry (Fv/Fm) and theeffective quantum yield of PSII (UPSII) in the lto1-2 mutant linewere lower than those of wild type. When the leaves were exposedto an irradiance of 1000 lmol m�2 s�1 for 1 h, these parameters de-creased sharply both in the lto1-2 mutant and the wild type. As thetime prolonged to 2 h, the values decreased continuously (Fig. 1aand b). The Fv/Fm ratio fell from 0.53 to 0.30, a 43% reduction, inthe lto1-2 mutant and from 0.84 to 0.70, a 17% reduction, in thewild type with 1 h of irradiance (Fig. 1a). Changes in the UPSII ratiofollowed a similar trend to the Fv/Fm ratio (Fig. 1b). As the highirradiance time was extended to 2 h, the decrease in photosyn-thetic activity was enhanced. The lto1-2 mutant was more sensitiveto strong light than the wild type, as was indicated by the morepronounced decrease in Fv/Fm and UPSII. This result suggestedthat the lto1-2 mutant suffered from severe photoinhibition.

To investigate whether photoinhibition in the lto1-2 mutantwas related to the energy dissipated through the xanthophyll cycleunder strong light conditions, a potent and widely used inhibitor ofVDE, DTT, was used [34,14]. Compared to the control treatment (noDTT), the reductions in Fv/Fm and UPSII were enhanced under highirradiance with the DTT treatment both in the lto1-2 mutant andthe wild type. The values of Fv/Fm and UPSII decreased as DTT con-centrations increased (Fig. 1a and b). The Fv/Fm ratio in the lto1-2mutant decreased more significantly than the wild type after DTTtreatment, suggesting that the lto1-2 mutant suffered from severephotoinhibition. The reduction in UPSII level in the wild type wasmore pronounced than for the lto1-2 mutant during exposure tohigh irradiance, especially after 1 h of treatment, indicating thatthe lto1-2 mutant was less sensitive to DTT than the wild type.Transgenic complementation ultimately recuperated the charac-

teristic changes described above (Tables S1–S2). Based on the re-sults above, it seems reasonable to assume that the aggravatedphotoinhibition observed in the lto1-2 mutant may be related tothe xanthophyll cycle. However, whether the suppression ofxanthophyll cycle was caused by inhibition of VDE needed to beinvestigated.

3.2. The inhibitor of VDE in the xanthophyll cycle alterednon-photochemical quenching (NPQ)

The xanthophyll cycle is an efficient thermal dissipation mech-anism in plants exposed to high light. The change in the amount ofthe de-epoxidated xanthophyll zeaxanthin is related to the extentof heat dissipation measured as NPQ.

Under growth light, the NPQ values were lower in the lto1-2mutant (0.86 ± 0.03) than in wild-type plants (1.2 ± 0.04)(Fig. 2a). After high irradiance for 1 h, the NPQ values increasedto 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 capabilityfor the dissipation of excess absorbed light energy.

The effects of DTT on NPQ under high irradiance were furtherinvestigated. Compared to the untreated control, the addition of3 mM DTT caused a significant decrease in NPQ in the wild typeand in the lto1-2 mutant after high irradiance for 1 h, as indicatedby the fact that the NPQ values fell from 1.72 to 0.96 in the wild-type and from 1.4 to 1.0 in the lto1-2 mutant (Fig. 2a). Increasing

Fig. 2. Changes in non-photochemical fluorescence quenching (NPQ) and the de-epoxidation state of xanthophyll cycle pigments. After 24 h in the dark, 1eaves werefed with 0 mM, 3 mM and 5 mM DTT at low light (30 lmol m�2 s�1) for 3 h at 22 �C.Leaves were then exposed to an irradiance of 1000 lmol m�2 s�1 for 1 h. (a) Thechange of non-photochemical quenching for the WT, lto1-2 and lto1-2C plants. lto1-2C represents transgenic complementation. 0 h indicates that the leaves wereplaced in growth light for 1 h as a control. (b) The change of the de-epoxidationstate. 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.

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the DTT concentration enhanced the reduction in NPQ values. Asimilar trend in NPQ values was observed both in transgenic com-plementation and wild type plants. Because the extent of thereduction in NPQ in the lto1-2 mutant was less than in the wildtype, the lto1-2 mutant had lower sensitivity to DTT than the wildtype. Then, the NPQ values for the lto1-2 mutant were slightlyhigher than the wild type after treatment with different concentra-tions of DTT.

3.3. The LTO1 protein was related to the de-epoxidation of thexanthophyll cycle

The great reduction in NPQ induced by DTT may be associatedwith a reduction in the de-epoxidation of the pigment interconver-sion within the xanthophyll cycle. We identified changes in theconcentrations of violaxanthin (Vio), antheraxanthin (Ant), andzeaxanthin (Zea) using high-performance liquid chromatography(HPLC). The de-epoxidation state of the xanthophyll cycle pig-ments 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 toVio. As the key enzyme of xanthophyll cycle, VDE can only be acti-vated by light and the light-driven transmembrane proton gradientto promote conversion of Vio to Zea via Ant. When leaves wereexposed to 1000 lmol m�2 s�1 irradiance for 1 h, the DEI valuesincreased both in the lto1-2 mutant and in the wild type, but the

DEI values were lower in the lto1-2 mutant line than in the wildtype and complementation plants. This result suggested that de-epoxidation of Vio to Zea via Ant was suppressed in the lto1-2mutant.

Although DTT is nonspecific inhibitor of VDE, the significantDTT-induced reduction in DEI observed in the lto1-2 mutant andwild type suggested that VDE was inhibited by DTT. Three millimo-lar DTT almost completely inhibited the formation of Zea. The DEIvalues were little difference among in the lto1-2 mutant, wild typeand complementation plants. Changes in the DEI were consistentwith changes in NPQ. The results above indicated that the LTO1protein was involved in regulation of the xanthophyll cycle, andthen regulating Zea-dependent energy dissipation in the photopro-tective mechanism.

3.4. The maximum efficiency of PSII photochemistry was affected whentreated with an inhibitor of protein synthesis in chloroplast

The previous investigation has shown that the redox activity ofLTO1 is indispensable for the assembly of PSII [27]. The D1 proteinlevel in PSII is significantly reduced in the lto1-2 mutant [28]. Theresults in Fig. 2a also showed that the NPQ values of the lto1-2 mu-tant were slightly higher than the wild type after treatment withdifferent concentrations of DTT under high light conditions. Thesedata suggested that there were other pathways of energy dissipa-tion in addition to the xanthophyll cycle being impacted by theLTO1 deficiency. The photoprotective pathway of D1 turnovermay be affected in the lto1-2 mutant. Previous work has shownthat the synthesis of D1 is not affected in the lto1-2 mutant [28];rather, the decrease in D1 protein concentration may be due to en-hanced degradation. To verify this hypothesis, SM, an inhibitor ofprotein synthesis in chloroplast, was used. As D1 protein is chloro-plast-encoded protein, so its synthesis is effectively prevented bySM [35].

Under growth light, the Fv/Fm ratio decreased in both the lto1-2mutant 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 de-cline in the wild type, and the difference in Fv/Fm between thelto1-2 mutant and wild type increased as the irradiation time in-creased (Fig. 3b). This result indicated that the lto1-2 mutant hada photodamage to PSII. Fig. 3c showed the changes in the ratio ofFv/Fm following exposure of leaves to strong light and treatmentwith SM. Compared with changes in the Fv/Fm ratio without SMtreatment, a more significant decrease in the Fv/Fm ratio was ob-served both in the lto1-2 mutant and wild type. After high irradi-ance treatment for 3 h following SM treatment, the Fv/Fm ratiodecreased to 0.02 in the lto1-2 mutant compared to 0.19 in the wildtype. To compare the decrease of Fv/Fm in wild type with that inlto1-2 mutant easily, the relative Fv/Fm ratio (the Fv/Fm ratio ofSM-treated leaves divided by the Fv/Fm ratio of control leaves)was compared by defining the initial values of Fv/Fm (before highirradiance) as 100% in the lto1-2 mutant and wild type. The per-centage of Fv/Fm was significantly lower in the lto1-2 mutant thanthat in the wild type (Fig. 3d), indicating that the lto1-2 mutant hada severe photodamage to PSII. Transgenic complementation ulti-mately demonstrated that a photodamage to PSII in the lto1-2 mu-tant plants was due to LTO1 (Tables S3–S4). These resultssuggested that LTO1 was involved in another photoprotectivepathway, D1 turnover.

3.5. The deficiency of LTO1 protein accelerated the D1 proteindegradation rate

To further investigate whether a severe photodamage to PSII inlto1-2 mutant was related to the photoprotective pathway of D1turnover, the quantity of D1 protein in the lto1-2 mutant, the wild

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 leaveswere 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 m�2 s�1

(growth light, a and b) or an irradiance of 1000 lmol m�2 s�1 (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.

72 Z.-B. Yu et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 68–75

type and complementation plants was analyzed by western blot.Recently research has shown that in lto1-2 mutants, D1 proteinhas only about half as much as in wild-type by using concentrationgradient of the sample proteins under growth light [28]. Our re-sults were consistent with previous investigation (Fig. 4a and b).Treatments with SM in low light (LL) for 3 h did not significantlyalter the D1 level in the lto1-2 mutant, wild type or complementa-tion plants (Fig. 4a and b).

After SM treatment, the leaves were exposed to a high light (HL)level of 1000 lmol m�2 s�1 for 3 h. D1 protein in the PSII centersinduced by high light levels was rapidly degraded. There were onlytrace amounts of D1 protein in the lto1-2 mutant and yet relativelyhigh quantities both in the wild type and compensation plants(Fig. 4a and b), indicating that there was more degradation of D1protein in the lto1-2 mutant than in the wild type. After treatmentwith SM and exposure to growth light (GL) for 3 h, Dl protein wasalso degraded both in the lto1-2 mutant and the wild type, butmuch less than that the degradation under high light with SMtreatment. However, the degradation of Dl protein in the lto1-2mutant was much more than that in the wild type and compensa-tion plants under this condition (Fig. 4a and b). Transgenic comple-mentation plants had even more D1 protein than the wild type,showing that D1 protein was overexpressed in complementationplants. Changes in the level of D1 under different illuminationintensities were consistent with changes in the Fv/Fm parameter,

suggesting that LTO1 protein deficiency accelerated the degrada-tion of D1 protein and affected the photoprotective pathway ofD1 turnover.

4. Discussion

4.1. Role of LTO1 in the regulation of energy dissipation depending onthe xanthophyll cycle

High light stress often inhibits photosynthesis and causes areduction in Fv/Fm and UPSII during the daytime in the summerseason [36]. In this investigation, the mutant lto1-2 line was shownto be more sensitive to high light (Fig. 1a and b). This result wasconsistent with previous research which has found that the lto1-1mutant has more severe photoinhibition [27]. In general,sensitivity to photoinhibition is thought to be governed by variousfactors including the ability to synthesize chemical energy, theefficiency of various mechanisms that dissipate excess excitationenergy to a harmless form and the capacity to repair photoinhibitedPSII during illumination [37].

A previous research has suggested that NPQ is higher in thelto1-1 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 varia-tion and it is hardly to get a solid conclusion. ‘‘It was not consis-tently higher or lower than wild-type leaves’’ as they mention in

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: streptomycinsulfate. a: GL: growth light with no SM. LL: low light for 3 h with 1 mM SM. GL: lowlight for 3 h with 1 mM SM and then growth light for 3 h. HL: low light for 3 h with1 mM SM and then high light for 3 h. b: Signals of immunoblots were quantifiedusing the ImageJ program. To compare D1 levels, ratios of WT were adjusted to 1under growth light and without SM treatment. The relative levels of D1 comparedto WT were calculated. The results were reported from three independentexperiments. 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 hwith 1 mM SM and then high light for 3 h.

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the article [27]. While our results obtained in the present studyhave demonstrated that the lto1-2 mutant had lower NPQ valuesthan that of wild type under growth light (120 lmol m�2 s�1)and high light (1000 lmol m�2 s�1) conditions for 1 h after 24 hdarkness by extensive experiments (Fig. 2a), suggesting that theability to dissipate excess absorbed light energy had been dimin-ished for lto1-2 mutant plants. When leaves were fed with DTT,the inhibitor of VDE in the xanthophyll cycle, NPQ values de-creased significantly in both the wild type and the lto1-2 mutantafter high irradiance for 1 h. Previous research has also shown thatthe lack of Zea could conceivably affect a Zea-dependent sustainedform of thermal energy dissipation when ascorbate is deficient andVDE is muted [38]. The lto1-2 mutant had lower sensitivity to DTTthan the wild type under high irradiance. Therefore, the contribu-tion of energy dissipation associated with the xanthophyll cyclewas less in the lto1-2 mutant than in the wild type under thiscondition.

HPLC analysis showed that the de-epoxidation state of pig-ments in the xanthophyll-cycle was also lower in the lto1-2 mutantthan in wild type under high irradiance (Fig. 2b), suggesting thatthe lto1-2 mutant had reduced xanthophyll cycle activity com-pared with the wild type. A significant DTT-induced decrease inthe DEI was observed both in the lto1-2 mutant and the wild typeunder high irradiance. Previous experiments have demonstratedthat DTT inhibits the HL-stimulated formation of zeaxanthin [39].Exogenous reduced glutathione (GSH) also markedly suppressthe formation of Zea via the xanthophyll cycle [40]. Consequently,the activity of VDE, the key enzyme in the xanthophyll cycle, isdependent on disulfide bond, while LTO1 is recently found to playa role in promoting the disulfide bond formation in the thylakoidlumen [27]. We inferred that the disulfide bond-promoting func-tion of LTO1 affected the activity of VDE and thus influenced thephotoprotective mechanism of the xanthophyll cycle.

According to the definition of transfer rates (ETR), ETR is deter-mined by UPSII and light density [41]. From our results, the lto1-2

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

4.2. LTO1 protein was involved in the process of Dl protein turnover

It is generally accepted that fast Dl turnover induced by highlight occurs in higher plants. This phenomenon has been demon-strated with radioactive label incorporation into Dl or degradationof relabeled protein [35]. Dl protein degradation and the insertionof newly synthesized Dl protein into the reaction center complex isclosely synchronized [46]. However, when the D1 protein degrada-tion rate exceeds the rate of synthesis, photoinhibition of PSII oc-curs [47]. Previous studies have shown that the synthesis of D1is not affected in the lto1-2 mutant both at the levels of transcrip-tion and translation in growth light. However, under this condition,the lto1-2 mutant has less D1 protein than wild type [28]. It waspossible that its degradation rate was higher in the lto1-2 mutantthan in wild-type plants. In this study, we used an inhibitor (SM)of protein synthesis in chloroplast, to focus on the degradation stepof the repair cycle of the PSII reaction center.

Our results showed that more intensive degradation of D1 pro-tein took place in the lto1-2 mutant than in the wild type (Fig. 4aand b). LTO1 protein is involved in the stability of PSII and can pro-mote the formation of disulfide bond in PsbO in vitro [27]. It is wellknown that the extrinsic PsbO requires intra-molecular disulfidebonds for its stabilization. Without the disulfide bonds probablyin the absence of LTO1 in the lto1-2 mutant, the PsbO cannot bindto the PSII core and thus may destabilize the whole PSII complex.Then the degradation of core protein D1of PSII may become moreeasily to occur. Consequently, the lack of LTO1 may be responsiblefor accumulation of small amounts of the D1 protein (that meansaccelerated degradation of the D1 protein) under light stress. Fur-thermore, deficiency of PsbD or PsbE in Chlamydomonas reinhardtiileads to a reduction in the level of D1, though transcript levels ofthe D1 synthesis are not affected [48,49]. A similar phenomenonis also found in Arabidopsis for the HCF243 protein. The deficiencyof this protein has no severe effects on the translation of D1 but ap-pears to accelerate its degradation instead [50]. In summary, LTO1protein deficiency affected the stability of PSII, the instability ofwhich facilitated the degradation of the D1 protein. This deficiencyeventually led to damage to the photoprotective pathway of D1turnover.

As to the relationship of xanthophyll cycle and D1 protein turn-over, some studies have demonstrated that the inactivation of PSIIreaction centers occurs as D1 turnover is inhibited, which depressthe xanthophyll cycle by affecting transmembrane pH gradient[51,52]. While the impairment of xanthophyll cycle can acceleratephotoinhibition in reverse by inhibiting the repair of photodam-aged D1 protein [53]. The results above were consistent with ourinvestigation. LTO1 protein was involved both photoprotectivemechanisms in the xanthophyll cycle and Dl protein turnover.

74 Z.-B. Yu et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 68–75

5. Abbreviations

Ant

antheraxanthin DEI de-epoxidation index DTT dithiothreitol UPSII effective quantum yield of PSII ETR electron transfer rate GL growth light qE ‘high-energy’ fluorescence quenching HPLC high-performance liquid chromatography HL high light LL low light LTO1 Lumen Thiol Oxidoreductase1 NPQ non-photochemical quenching OEC oxygen evolving complex PSII photosystem II PSI photosystem I LHCII photosystem II light harvesting complex FMS-2 pulse-modulated fluorometer PFD photon flux density PAGE polyacrylamide gel electrophoresis pH potential of hydrogen D1 reaction center protein of PSII ROS reactive oxygen species SM streptomycin sulfate SDS sodium dodecyl sulfate Fv/Fm the maximum efficiency of PSII photochemistry Vio violaxanthin VKOR vitamin K epoxide reductase VDE violaxanthin de-epoxidase Zea zeaxanthin

Acknowledgement

This work was supported by Special Research Fund of PublicWelfare of China Agricultural Ministry (201303093).

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.jphotobiol.2013.11.003.

References

[1] J. Barber, B. Andersson, Too much of a good thing: light can be bad forphotosynthesis, Trends Biochem. Sci. 17 (1992) 61–66.

[2] Y. Nishiyama, H. Yamamoto, S.I. Allakhverdiev, M. Inaba, A. Yokota, N. Murata,Oxidative stress inhibits the repair of photodamage to the photosyntheticmachinery, EMBO J. 20 (2001) 5587–5594.

[3] K. Apel, H. Hirt, Reactive oxygen species: metabolism, oxidative stress, andsignal transduction, Annu. Rev. Plant Biol. 55 (2004) 373–399.

[4] E.M. Aro, I. Virgin, B. Andersson, Photoinhibition of photosystem II.Inactivation, protein damage and turnover, Biochim. Biophys. Acta 1143(1993) 113–134.

[5] A. Krieger-Liszkay, C. Fufezan, A. Trebst, Singlet oxygen production inphotosystem II and related protection mechanism, Photosynth. Res. 98(2008) 551–564.

[6] E.H. Murchie, K.K. Niyogi, Manipulation of photoprotection to improve plantphotosynthesis, Plant Physiol. 155 (2011) 86–92.

[7] S. Takahashi, M.R. Badger, Photoprotection in plants: a new light onphotosystem II damage, Trends Plant Sci. 16 (2011) 53–60.

[8] P. Muller, X.P. Li, K.K. Niyogi, Non-photochemical quenching. A response toexcess light energy, Plant Physiol. 125 (2001) 1558–1566.

[9] M. Havaux, K.K. Niyogi, The violaxanthin cycle protects plants fromphotooxidative damage by more than one mechanism, Proc. Natl. Acad. Sci.USA 96 (1999) 8762–8767.

[10] P. Jahns, A.R. Holzwarth, The role of the xanthophyll cycle and of lutein inphotoprotection of photosystem II, Biochim. Biophys. Acta 2012 (1817) 182–193.

[11] P. Muller-Moule, P.L. Conklin, K.K. Niyogi, Ascorbate deficiency can limitviolaxanthin de-epoxidase activity in vivo, Plant Physiol. 128 (2002) 970–977.

[12] A.D. Hieber, R.C. Bugos, A.S. Verhoeven, H.Y. Yamamoto, Overexpression ofviolaxanthin de-epoxidase: properties of C-terminal deletions on activity andpH-dependent lipid binding, Planta 214 (2002) 476–483.

[13] K.K. Niyogi, A.R. Grossman, O. Bjorkman, Arabidopsis mutants define a centralrole for the xanthophyll cycle in the regulation of photosynthetic energyconversion, Plant Cell 10 (1998) 1121–1134.

[14] J.-L. Huang, L.-L. Cheng, Z.-X. Zhang, Molecular cloning and characterization ofviolaxanthin de-epoxidase (VDE) in Zingiber officinale, Plant Sci. 172 (2007)228–235.

[15] M. Hall, A. Mata-Cabana, H.-E. Åkerlund, F.J. Florencio, W.P. Schröder, M.Lindahl, T. Kieselbach, Thioredoxin targets of the plant chloroplast lumen andtheir implications for plastid function, Proteomics 10 (2010) 987–1001.

[16] J.A. Raven, The cost of photoinhibition, Physiol. Plant 142 (2011) 87–104.[17] M. Edelman, A.K. Mattoo, D1-protein dynamics in photosystem II: the

lingering enigma, Photosynth. Res. 98 (2008) 609–620.[18] P.J. Nixon, F. Michoux, J. Yu, M. Boehm, J. Komenda, Recent advances in

understanding the assembly and repair of photosystem II, Ann. Bot. 106 (2010)1–16.

[19] E. Baena-Gonzalez, E.M. Aro, Biogenesis, assembly and turnover ofphotosystem II units, Philos. Trans. R. Soc. Lond. B Biol. Sci. 357 (2002)1451–1459 (discussion 1459–1460).

[20] E.M. Aro, M. Suorsa, A. Rokka, Y. Allahverdiyeva, V. Paakkarinen, A. Saleem, N.Battchikova, E. Rintamaki, Dynamics of photosystem II: a proteomic approachto thylakoid protein complexes, J. Exp. Bot. 56 (2005) 347–356.

[21] Y. Kato, W. Sakamoto, Protein quality control in chloroplasts: a current modelof D1 protein degradation in the photosystem II repair cycle, J. Biochem. 146(2009) 463–469.

[22] S. Takahashi, N. Murata, How do environmental stresses acceleratephotoinhibition?, Trends Plant Sci 13 (2008) 178–182.

[23] M. Bertamini, N. Nedunchezhian, Photoinhibition and recovery ofphotosynthesis in leaves of Vitis berlandieri and Vitis rupestris, J. PlantPhysiol. 161 (2004) 203–210.

[24] K. Kojima, M. Oshita, Y. Nanjo, K. Kasai, Y. Tozawa, H. Hayashi, Y. Nishiyama,Oxidation of elongation factor G inhibits the synthesis of the D1 protein ofphotosystem II, Mol. Microbiol. 65 (2007) 936–947.

[25] Y. Nishiyama, S.I. Allakhverdiev, N. Murata, A new paradigm for the action ofreactive oxygen species in the photoinhibition of photosystem II, Biochim.Biophys. Acta 1757 (2006) 742–749.

[26] W.K. Feng, L. Wang, Y. Lu, X.Y. Wang, A protein oxidase catalysing disulfidebond formation is localized to the chloroplast thylakoids, FEBS J. 278 (2011)3419–3430.

[27] M. Karamoko, S. Cline, K. Redding, N. Ruiz, P.P. Hamel, Lumen thioloxidoreductase1, a disulfide bond-forming catalyst, is required for theassembly of photosystem II in Arabidopsis, Plant Cell 23 (2011) 4462–4475.

[28] Y. Lu, H.R. Wang, H. Li, H.R. Cui, Y.G. Feng, X.Y. Wang, A chloroplast membraneprotein LTO1/AtVKOR involving in redox regulation and ROS homeostasis,Plant Cell Rep. (2013).

[29] L.T. Zhang, Z.S. Zhang, H.Y. Gao, X.L. Meng, C. Yang, J.G. Liu, Q.W. Meng, Themitochondrial alternative oxidase pathway protects the photosyntheticapparatus against photodamage in Rumex K-1 leaves, BMC Plant Biol. 12(2012) 40.

[30] D.M. Pandey, K.-H. Kang, U.-D. Yeo, Effects of excessive photon on thephotosynthetic pigments and violaxanthin de-epoxidase activity in thexanthophyll cycle of spinach leaf, Plant Sci. 168 (2005) 161–166.

[31] L. Peng, J. Ma, W. Chi, J. Guo, S. Zhu, Q. Lu, C. Lu, L. Zhang, Low PSIIAccumulation1 is involved in efficient assembly of photosystem II inArabidopsis thaliana, Plant Cell 18 (2006) 955–969.

[32] D.I. Arnon, Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Betavulgaris, Plant Physiol. 24 (1949) 1–15.

[33] Y. Yamamoto, Y. Nishi, H. Yamasaki, S. Uchida, S. Ohira, Assay ofphotoinhibition of photosystem II and protease activity, Methods Mol. Biol.274 (2004) 217–227.

[34] Y. Yin, S. Li, W. Liao, Q. Lu, X. Wen, C. Lu, Photosystem II photochemistry,photoinhibition, and the xanthophyll cycle in heat-stressed rice leaves, J. PlantPhysiol. 167 (2010) 959–966.

[35] B. Schnettger, C. Critchley, U.J. Santore, M. Graf, G.H. Krause, Relationshipbetween photoinhibition of photosynthesis, D1 protein turnover andchloroplast structure: effects of protein synthesis inhibitors, Plant CellEnviron. 17 (1994) 55–64.

[36] N. Murata, S. Takahashi, Y. Nishiyama, S.I. Allakhverdiev, Photoinhibition ofphotosystem II under environmental stress, Biochim. Biophys. Acta 1767(2007) 414–421.

[37] E. Tyystjarvi, K. Ali-Yrkko, R. Kettunen, E.M. Aro, Slow degradation of the d1protein is related to the susceptibility of low-light-grown pumpkin plants tophotoinhibition, Plant Physiol. 100 (1992) 1310–1317.

Z.-B. Yu et al. / Journal of Photochemistry and Photobiology B: Biology 130 (2014) 68–75 75

[38] P. Muller-Moule, M. Havaux, K.K. Niyogi, Zeaxanthin deficiency enhances thehigh light sensitivity of an ascorbate-deficient mutant of Arabidopsis, PlantPhysiol. 133 (2003) 748–760.

[39] R. Goss, C. Opitz, B. Lepetit, C. Wilhelm, The synthesis of NPQ-effectivezeaxanthin depends on the presence of a transmembrane proton gradient anda slightly basic stromal side of the thylakoid membrane, Planta 228 (2008)999–1009.

[40] C.C. Xu, L. Li, T. Kuang, The inhibited xanthophyll cycle is responsible for theincrease in sensitivity to low temperature photoinhibition in rice leaves fedwith glutathione, Photosynth. Res. 65 (2000) 107–114.

[41] J.E. Clarke, G.N. Johnson, In vivo temperature dependence of cyclic andpseudocyclic electron transport in barley, Planta 212 (2001) 808–816.

[42] S. de Bianchi, L. Dall’Osto, G. Tognon, T. Morosinotto, R. Bassi, Minor antennaproteins CP24 and CP26 affect the interactions between photosystem IIsubunits and the electron transport rate in grana membranes of Arabidopsis,Plant Cell 20 (2008) 1012–1028.

[43] P. Horton, M.P. Johnson, M.L. Perez-Bueno, A.Z. Kiss, A.V. Ruban,Photosynthetic acclimation: does the dynamic structure and macro-organisation of photosystem II in higher plant grana membranes regulatelight harvesting states?, FEBS J 275 (2008) 1069–1079.

[44] D.M. Kramer, T.J. Avenson, G.E. Edwards, Dynamic flexibility in the lightreactions of photosynthesis governed by both electron and proton transferreactions, Trends Plant Sci. 9 (2004) 349–357.

[45] T. Shikanai, Y. Munekage, K. Kimura, Regulation of proton-to-electronstoichiometry in photosynthetic electron transport: physiological function inphotoprotection, J. Plant Res. 115 (2002) 0003–0010.

[46] B. Andersson, J. Barber, Mechanisms of photodamage and protein degradationduring photoinhibition of photosystem II, in: N. Baker (Ed.), Photosynthesisand the Environment, Springer, Netherlands, 2004, pp. 101–121.

[47] S. Takahashi, N. Murata, How do environmental stresses acceleratephotoinhibition?, Trends Plant Sci 13 (2008) 178–182.

[48] J.M. Erickson, M. Rahire, P. Malnoe, J. Girard-Bascou, Y. Pierre, P. Bennoun, J.D.Rochaix, Lack of the D2 protein in a Chlamydomonas reinhardtii psbD mutantaffects photosystem II stability and D1 expression, EMBO J. 5 (1986) 1745–1754.

[49] F. Morais, J. Barber, P.J. Nixon, The chloroplast-encoded alpha subunit ofcytochrome b-559 is required for assembly of the photosystem two complexin both the light and the dark in Chlamydomonas reinhardtii, J. Biol. Chem. 273(1998) 29315–29320.

[50] D. Zhang, G. Zhou, B. Liu, Y. Kong, N. Chen, Q. Qiu, H. Yin, J. An, F. Zhang, F.Chen, HCF243 encodes a chloroplast-localized protein involved in the D1protein stability of the arabidopsis photosystem II complex, Plant Physiol. 157(2011) 608–619.

[51] C.-D. Jiang, H.-Y. Gao, Q. Zou, Enhanced thermal energy dissipation dependingon xanthophyll cycle and D1 protein turnover in iron-deficient maize leavesunder high irradiance, Photosynthetica 39 (2001) 269–274.

[52] A.W. Russell, C. Critchley, S.A. Robinson, L.A. Franklin, G. Seaton, W.S. Chow,J.M. Anderson, C.B. Osmond, Photosystem II regulation and dynamics of thechloroplast D1 protein in arabidopsis leaves during photosynthesis andphotoinhibition, Plant Physiol. 107 (1995) 943–952.

[53] S. Takahashi, S.E. Milward, D.Y. Fan, W.S. Chow, M.R. Badger, How does cyclicelectron flow alleviate photoinhibition in Arabidopsis?, Plant Physiol 149(2009) 1560–1567.