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Jia-Jia Du
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Ying Lu
Hao-Ran Cui

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ORIGINAL RESEARCH
published: xx April 2015
doi: 10.3389/fpls.2015.00238
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Q1 018

The conservative cysteines in

transmembrane domain of
AtVKOR/LTO1 are critical for
photosynthetic growth and
photosystem II activity in Arabidopsis

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Jia-Jia Du , Chun-Yan Zhan , Ying Lu , Hao-Ran Cui and Xiao-Yun Wang *

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State Key Laboratory of Crop Biology, College of Life Science, Shandong Agricultural University, Taian, China

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Edited by:
Richard Sayre,
New Mexico Consortium at Los
Alamos National Labs, USA
Reviewed by:
Xinguang Zhu,
Chinese Academy of Sciences, China
Wei Huang,
Kunming Institute of Botany
Chinese Academy of Sciences, China
*Correspondence:
Xiao-Yun Wang,
State Key Laboratory of Crop Biology,
College of Life Science, Shandong
Agricultural University,
Daizong Street 61, Taian,
Shandong 271018, China
xyunwang@sdau.edu.cn
Specialty section:
This article was submitted to Plant
Physiology, a section of the journal
Frontiers in Plant Science
Received: 08 October 2014
Accepted: 25 March 2015
Published: xx April 2015
Citation:
Du J-J, Zhan C-Y, Lu Y, Cui H-R and
Wang X-Y (2015) The conservative
cysteines in transmembrane domain
of AtVKOR/LTO1 are critical for
photosynthetic growth and
photosystem II activity in Arabidopsis.
Front. Plant Sci. 6:238.
doi: 10.3389/fpls.2015.00238

Thylakoid protein vitamin K epoxide reductase (AtVKOR/LTO1) is involved in

oxidoreduction. The deficiency of this compound causes pleiotropic defects in
Arabidopsis thaliana, such as severely stunted growth, smaller sized leaves, and
delay of flowering. Transgenic complementation of wild-type AtVKOR (VKORWT ) to
vkor mutant lines ultimately demonstrates that the phenotype changes are due to
this gene. However, whether AtVKOR functions in Arabidopsis through its protein
oxidoreduction is unknown. To further study the redox-active sites of AtVKOR in
vivo, a series of plasmids containing cysteine-mutant VKORs were constructed and
transformed into vkor deficient lines. Compared with transgenic AtVKORWT plants, the
size of the transgenic plants with a single conservative cysteine mutation (VKOR C109A ,
VKORC116A , VKORC195A , and VKORC198A ) were smaller, and two double-cysteine
mutations (VKORC109AC116A and VKORC195AC198A ) showed significantly stunted growth,
similar with the vkor mutant line. However, mutations of two non-conservative cysteines
(VKORC46A and VKORC230A ) displayed little obvious changes in the phenotypes of
Arabidopsis. Consistently, the maximum and actual efficiency of photosystem II (PSII)
in double-cysteine mutation plants decreased significantly to the level similar to that
of the vkor mutant line both under normal growth light and high light. A significantly
decreased amount of D1 protein and increased accumulation of reactive oxygen species
were observed in two double-cysteine mutations under high light. All of the results above
indicated that the conservative cysteines in transmembrane domains were the functional
sites of AtVKOR in Arabidopsis and that the oxidoreductase activities of AtVKOR were
directly related to the autotrophic photosynthetic growth and PSII activity of Arabidopsis
thaliana.

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Keywords: AtVKOR, cysteine, disulfide bond, photosystem II, D1 protein

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Abbreviations: AtVKORCXXA/AXXC/AXXA plants, complemented cysteine-mutant VKORs/vkor plants; AtVKORWT plants,

complemented wild-type AtVKOR/vkor plants; H2 O2, hydrogen peroxide; LTO1, Lumen thiol oxidoreductase 1; NPQ, nonphotochemical quenching; O2 , superoxide anion radical; PSII, actual PSII eciency; PSII, photosystem II; qRT-PCR,
quantitative reverse transcription-polymerase chain reaction; ROS, reactive oxygen species; VDE, violaxanthin de-epoxidase;
VKOR, vitamin K epoxide reductase; WT, wild-type Arabidopsis.

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The role of conservative cysteines of AtVKOR in Arabidopsis

Introduction

oxygen-evolving complex (Roose et al., 2010; Roberts et al.,

2012). FKBP13, a peptidyl-prolyl cistrans isomerase in the thylakoid lumen also contains an essential disulde bond and its
sulfhydryl oxidation plays a vital role in the photosynthetic electron transport chain (Gopalan et al., 2004; Kang et al., 2008).
Experiments reveal that another luminal protein regulated by
AtVKOR may be VDE, a luminal enzyme involved in thermal dissipation through the xanthophyll cycle, the activity of
which is also dependent on its sulfhydryl oxidation (Bugos and
Yamamoto, 1996; Latowski et al., 2004; Yu et al., 2014). Since
the redox regulation mechanism of chloroplasts in high plants
is complex and vague, we wondered whether AtVKOR regulated their growth and development through its oxidoreductase
activity.
In this investigation, plasmids containing cysteine-mutant
VKORs were constructed and transformed into vkor mutant
lines. Based on the stunted growth phenotype and decreased
PSII activity of vkor homozygosities containing mutations of
single/double conservative cysteines in the VKOR domain, the
important role of the oxidoreductase activities of AtVKOR in
photosynthetic growth and PSII activity was conrmed.

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In chloroplasts, disulde bond formation, a covalent bond

between two cysteines, is crucial for the maturation and function of proteins and other organelles (Jocelyn, 1967; Buchanan
and Luan, 2005; Onda, 2013). The majority of protein disuldes
in chloroplasts are considered to be one important inert covalent linkage for structure stabilizing modications (Nagahara,
2011; Romero et al., 2014). However, some regulatory disulde
bonds serve as signaling elements by interchanging their reduced
or oxidized states, which plays important roles in photosynthesis, gene expression, signal transduction, and stress resistance
(Buchanan and Luan, 2005; Wouters et al., 2010; Cremers and
Jakob, 2013; Karamoko et al., 2013; Kieselbach, 2013). Intensive
studies regarding the enzymatic reduction of disulde bonds in
redox-regulated proteins in chloroplasts have been conducted,
while the reversible process, the formation of a disulde bond, has
yet to be elucidated (Aro and Ohad, 2003; Buchanan and Luan,
2005; Hall et al., 2010; Cremers and Jakob, 2013; Karamoko et al.,
2013; Kieselbach, 2013).
A novel thylakoid protein, VKOR, from Cyanobacteria and
Arabidopsis thaliana has been identied to promote disulde
bond formation (Furt et al., 2010; Li et al., 2010; Feng et al.,
2011; Karamoko et al., 2011). Compared with Cyanobacteria
VKOR, plant VKOR has an additional transit peptide at the Nterminus that targets the protein to chloroplasts (Furt et al., 2010;
Feng et al., 2011; Wan et al., 2014). Dierent from VKOR in
mammals, both thylakoid VKORs are fusion proteins comprising two domains, a transmembrane/VKOR domain and a soluble thioredoxin-like/Trx-like domain (Oldenburg et al., 2006; Li
et al., 2010; Feng et al., 2011). In Arabidopsis, the transmembrane
domain/VKOR domain of AtVKOR, which is homologous to
VKORs from mammalian, Synechococcus sp. and Mycobacterium
tuberculosis, contains two non-conservative cysteines (Cys46,
Cys230) and four conservative cysteines forming two pairs (Feng
et al., 2011). One pair is in a separated form (Cys109, Cys116),
and the other pair is in a canonical Cys-X-X-Cys motif (Cys195,
Cys198). Functional analyses reveal that these two pairs of conservative cysteines are indispensable for the oxidoreductase activities of AtVKOR in the process of catalyzing disulde bond
formation in Escherichia coli (Feng et al., 2011; Karamoko et al.,
2011). In M. tuberculosis, two pairs of conservative cysteines of
MtbVKOR are also found to play critical roles in the formation of
disulde bonds (Wang et al., 2011).
Arabidopsis VKOR is also called LTO1, and mutant lines
of vkor (also called lto1) display severely decient photosynthetic growth and low activities of PSII (Karamoko et al.,
2011; Lu et al., 2013). Interestingly, the underlying mechanism of defects in the vkor mutant line has not been determined, although transgenic VKORWT to vkor mutant lines
demonstrates that the phenotype changes are due to this gene
(Karamoko et al., 2011; Lu et al., 2013). In vitro, the recombinant Trx-like domain of AtVKOR can promote the disulde bond formation of targets in chloroplasts, such as proteins
PsbO and FKBP13 (Karamoko et al., 2011; Lu et al., 2013).
PsbO, a luminal subunit of PSII, carries a single intramolecular disulde bond and is essential for the stability of the

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Materials and Methods

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Plant Materials and Growth Conditions

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Wild-type Arabidopsis ecotype Columbia and the T-DNA insertion vkor mutant line have been described in a previous work (Lu
et al., 2013). For growth on Murashige and Skoog (MS) medium,
the seeds of wild-type and transgenic plants were surfacesterilized with 70% ethanol and 2.6% bleach for 5 and 10 min,
respectively. Then, seeds were washed more than ve times
with sterilized water containing detergent Tween-20. The washed
seeds of transgenic or wild-type plants (WTs) were allowed to germinate on MS medium with or without 50 g ml1 kanamycin.
The seeds on plates were stratied for 48 h at 4 C in the dark
for synchronized germination. After 2 weeks, the plantlets were
transplanted into vermiculite under 120 mol m2 s1 with
short-day conditions (8-h-light/16-h-dark) or long-day conditions (16-h-light/8-h-dark) at a constant temperature of 22 C.

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Plasmids for Plant Transformation

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The veried sequences of single/double cysteine-mutant

AtVKORs were fused to plant transformation plasmid
pBI121, following the previous operations of the plasmid
pBI121 of wild-type AtVKOR with full-length cDNA (Lu
et al., 2013). The heterozygotic Arabidopsis of wild-type
Columbia and the vkor mutant line were determined by
T-DNA specic primers (Forward primer of AtVKOR, 5 CTTACCTGCAATGCAATGTTG-3; reverse primer of
AtVKOR, 5 -ACCAGTTTCCAATTCGTGATG-3; T-DNA
specic primer, 5 -GCGTGGACCGCTTGCTGCAACT-3) and
were used for oral dip transformation. Transgenic plants were
selected for kanamycin resistance and veried by genomic
PCR with specic primers (forward primer for genomic PCR,
5 -GGCCATGGAGTCAAAGATTC-3; reverse primer for
genomic PCR, 5 -CATTGCAGTCGTGATCCC-3). In the next

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The role of conservative cysteines of AtVKOR in Arabidopsis

generation, the homozygotes of cysteine-mutant VKORs to vkor

mutant background were screened by T-DNA specic primers as
described above.

D1 amount was quantied by assaying the intensity in the western

blot using Image J software.

H2 O2 and O2 Determination

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The content of H2 O2 and O2 was determined according to

the previous method (Jiang and Zhang, 2002; Clore et al., 2008).
Leaves of the vkor mutant line, cysteine-mutant, VKORs and
VKORWT transgenic plants under normal growth light and
after 2-h high light treatment were ground to a ne power
in liquid nitrogen and extracted using 50 mM PBS (pH 7.8).
The absorbance was determined at 436 and 530 nm, respectively, and the contents of H2 O2 and O2 were calculated
according to the standard curve of H2 O2 reagent and NaNO2
reagent. Each experiment was carried out in three biological
replicates.

RNA Extraction and Semiquantitative

RT-PCR
The leaves of WTs, the vkor mutant line, and transgenic cysteinemutant VKORs and AtVKORWT plants were used for total RNA
isolation using the method described by Lu et al. (2014). The
cDNA synthesis was performed according to standard procedures of RevertAid Fist Strand cDNA Synthesis Kit (Fermentas,
Canada). In a semiquantitative RT-PCR assay, elongation factor
1-alpha (EF1-) was used as a control for normalization. The
PCR cycles were as follows: one cycle of 5 min at 95 C, followed by 28 cycles each of 30 s at 95 C, 30 s at 57 C, and 30 s
at 72 C, nal cycle of 8 min at 72 C. Forward primer of EF1-,
5 -GAGGCTGGTATCTCTAAGGA-3, reverse primer of EF1, 5 -GGAAGTGCCTCAAGAAGAGA-3; forward primer of
AtVKOR, 5 -GTCGGTAACTTCTTATCCTAGACG-3, reverse
primer of AtVKOR, 5 - CTGAGAGTTTTGTGCTAAGG-3.
Each reaction was carried out in three biological replicates.

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Replacement of the Conservative Cysteines

in AtVKOR Caused the Pleiotropic Growth
Defects in Arabidopsis

Measurements of Chlorophyll Fluorescence

under Different Light

Six cysteines exist in the transmembrane domain of AtVKOR,

including two non-conservative cysteines (Cys46, Cys230) and
four conservative cysteines forming two pairs (Cys109/Cys116
and Cys195/Cys198; Feng et al., 2011). Single/double-cysteine(s)
mutant VKORs and wild-type AtVKOR (VKORWT ) were successfully expressed in vkor mutant lines, respectively (Figure 1B).
The representative phenotypes of the vkor homozygosities
with the insertion of cysteine-mutant VKORs are shown in
Figure 1A. While the AtVKORWT transgenic plants can completely recover the phenotype defects in the vkor line, transgenic plants with a single conservative cysteine mutation
(AtVKORC109A , AtVKORC116A , AtVKORC195A , AtVKORC198A )
did only partly recover the phenotype defects in the vkor
mutant lines (Figure 1A). When double conservative cysteines were mutated to alanines, the AtVKORC109AC116A and
AtVKORC195AC198A transgenic plants completely lost the ability to compensate for these defects in the vkor mutant, displaying signicantly stunted growth, smaller sized leaves, and
delayed owering, quite similar to the defects of the vkor mutant
line. However, the transgenic plants with non-conservative cysteine mutations (AtVKORC46A , AtVKORC230A ) showed no obvious dierence, compared with WT plants and AtVKORWT
plants.
The changes of biomass were further detected in the transgenic plants. The fresh weight (FW) of transgenic plants
with double-cysteine mutations (AtVKORC109AC116A and
AtVKORC195AC198A ) decreased signicantly, only about 30% of
that of WT and a little more than that of vkor decient lines
(Figure 2). As to transgenic plants with the non-conservative
cysteine mutations (AtVKORC46A and AtVKORC230A ), the
FW decreased a little, about 95% of that of WTs (Figure 2).
The changes of biomass were consistent with the phenotypes
observed above, further conrming indispensability of the
conservative cysteines of AtVKOR in photosynthetic growth of
plants.

Fully expanded leaves from 8-weeks-old plants were detached

and incubated in sterilized water under normal growth light
(120 mol m2 s1 ) and high light stress (600 mol m2 s1 ) for
2 h, respectively. Chlorophyll uorescence was measured using a
pulsemodulated uorometer (FMS-2, Hansatech, UK) as previously described (Yu et al., 2014). One part of the treated leaves
were shielded for dark-adaption for more than 15 min, and then,
the dark adapted leaves were used for the measurement of maximum quantum yield of PSII (Fv/Fm; Fv, the variable chlorophyll
uorescence yield, dened as Fm-Fo). The other part of treated
leaves were directly used for the measurement of the  PSII. The
parameters were then calculated as previously described (Krause
and Weis, 1991 ): Fv/Fm = (Fm Fo)/Fm, PSII = (Fm
Fs)/Fm, and NPQ = (FmFm)/Fm. In every experiment, at least
six leaves were measured, and three independent experiments
were conducted.

Thylakoid Membrane Protein Preparation

and Western Blot Detection
According to the previous description, the thylakoid membranes were prepared from the leaves of the vkor mutant line,
cysteine-mutant AtVKORs and AtVKORWT transgenic plants
under normal growth light or 2-h high light treatment (Lu
et al., 2013; Yu et al., 2014). The chlorophyll content was determined in 80% (v/v) acetone according to previous operations (Yu
et al., 2014). Protein samples corresponding to equal amounts
of chlorophyll were separated through 15% sodium dodecyl
sulfatepolyacrylamide gel electrophoresis (SDSPAGE). Then,
the bands of proteins were transferred onto immobilon-P membranes (Millipore, USA) and blotted with specic D1-antibody.
The immune-decorated signals were detected by sensitive uorography with enhanced chemiluminescence (Amersham, Japan).
More than three independent experiments were conducted. The

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FIGURE 1 | Characteristics of cysteine-mutant AtVKORs plants. (A) The phenotypes of cysteine-mutant AtVKORs plants; plants were grown on soil for
8 weeks under normal growth conditions. (B) Transcriptional level analysis of AtVKOR in plants of cysteine-mutant AtVKORs.

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slight dierence exists. The mutations of two non-conservative

cysteines does not aect the function of AtVKOR in catalyzing the formation of a disulde bond, but each single or double
mutation of conservative cysteines absolutely leads to the loss of
the catalytic ability by checking the motility and -galactosidase
activity in E. coli (Feng et al., 2011). Unlike the phenotype changes
of bacteria, in Arabidopsis, transgenic plants with a single conservative cysteine VKORs mutation partly recovered the decient
phenotypes, which revealed that a single cysteine mutation did
not break down the electron transferring in plants (Figure 1).
Among the four single mutations, the growth of AtVKORC195A
was the worst. Combined with the results of double mutations, we
supposed the cysteine 195 was directly involved in electron transferring. Characterizations of plant phenotypes demonstrated that
all four conservative cysteines could form in pairs when AtVKOR
played functions in photosynthetic growth in Arabidopsis.

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PSII Activities were Inhibited in the

Transgenic Plants with Conservative
Cysteine VKORs Mutation Both under
Normal Growth Light and High Light

FIGURE 2 | The biomass of cysteine-mutant AtVKORs plants. The fresh

weights (FW) of up-ground parts of WT, vkor mutant, AtVKORWT, and
cysteine-mutant VKORs plants were measured. The experiments were
repeated at least three times and more than three plants were used each time.
The error bars indicated the SD. Significant differences are determined using
one-way ANOVA and Ducans Multiple Range Test, as indicated with different
letters at P < 0.05 significance level.

Arabidopsis VKOR has been proven to be required for the assembly of PSII; Karamoko et al., 2011), we wondered whether the
cysteines in AtVKOR also aected the activities of PSII. The
maximal quantum yield of PSII (Fv/Fm), an indicator for the eciency of PSII photochemistry (Lu et al., 2011), was determined
by a chlorophyll uorescence measurement in the WT plants,
vkor mutant line, cysteine-mutant VKORs, and AtVKORWT

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The eects of the non-conservative and conservative cysteines

of AtVKOR to Arabidopsis phenotypes were consistent with their
eects to the formation of a disulde bond in E. coli, although a

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The role of conservative cysteines of AtVKOR in Arabidopsis

transgenic plants under growth light (120 mol m2 s1 ). As

shown in Table 1, no signicant dierence in the Fv/Fm ratio was
observed among the AtVKORC46A , AtVKORC230A , AtVKORWT
transgenic plants, and WT plants (Table 1), indicating that wildtype VKOR as well as the mutation of non-conservative cysteines
in the VKOR domain were all sucient to restore photosynthetic eciency under normal growth conditions. However, the
ratios of Fv/Fm in AtVKORC109AC116A and AtVKORC195AC198A
plants (respectively, 0.662 and 0.673) were dramatically lower
than that in the AtVKORWT plant (averagely 0.866), indicating
the decreased activity of the reaction centers of PSII due to the
redox-inactive mutation of conservative cysteines in the VKOR
domain. Moreover, a single mutation at Cys195 could also arouse
a signicant decrease in Fv/Fm (averagely 0.699). A similar trend
of actual PSII photochemical eciency (PSII) was observed
in the investigated plants (Table 1). These low chlorophyll uorescence parameters in transgenic plants with mutations of
conservative cysteine VKORs were correlated with the impaired
photosynthesis and reduced activities of PSII.
Excess light has negative impacts on plant photosynthesis, and
previous research shows that the mutant line of vkor is sensitive
to high light (Yu et al., 2014). Our results demonstrated that high
irradiance (HL, 600 mol m2 s1 for 2 h) increased the dierences on Fv/Fm and PSII among double conservative cysteinemutant VKORs and AtVKORWT plants, compared with normal
growth light (GL, 120 mol m2 s1 ), as shown in Table 1,
suggesting that the photoinhibition in AtVKORC109AC116A and
AtVKORC195AC198A plants was further aggravated when exposed
to high light.
To avoid photodamage, photosynthetic organisms have developed various photoprotective mechanisms to resist photooxidative damage and to repair damaged protein components
(Nishiyama et al., 2001; Murata et al., 2007). One important
pathway is to minimize excitation pressure on PSII by thermal dissipation, which can be reected by the value of NPQ.
In this investigation, we found that the NPQ values were lower

in the AtVKORC109AC116A (averagely 0.945, GL; averagely 1.112,

HL) and AtVKORC195AC198A (averagely 0.949, GL; averagely
1.116, HL) plants compared with AtVKORWT (averagely 1.201,
GL; averagely 1.612, HL) plants under dierent illumination
(Table 1), suggesting a low capability in the dissipation of excess
light. Previous investigations have proved that the npq1 mutant
exhibits greatly reduced NPQ with decient VDE, a vital enzyme
in xanthophyll cycle (Niyogi et al., 1998; Han et al., 2010).
AtVKOR/LTO1 has been proven to be related with the xanthophyll cycle, one important mechanism to dissipate excess
thermal energy (Yu et al., 2014). We supposed that mutations of
conservative cysteines of AtVKOR aected the cycle of xanthophyll and resulted in the impaired photoprotection for thermal
dissipation.

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The turnover of D1 protein is one of photoprotective processes

in PSII under high light stress (Nishiyama et al., 2001; Huesgen
et al., 2006). Under normal growth light, the levels of D1 protein in transgenic plants with mutant AtVKORs of conservative
cysteine were decreased, especially in double-cysteine mutants
AtVKORC109AC116A and AtVKORC195AC198A (Figures 3 and 4).
High irradiance increased the decrease extent of D1 protein, and
only trace amount of D1 accumulation could be detected in the
vkor mutant line and double-cysteine mutant plants, which is
consistent with the previous result that the deciency of AtVKOR
accelerates the degradation of D1 protein (Yu et al., 2014). Little
dierence was observed in the level of D1 protein among the
transgenic plants with mutant AtVKORs of non-conservative
cysteine and AtVKORWT (Figures 3 and 4). The results above
suggested that the turnover of D1 protein in the repair of photodamaged were impaired due to the mutations of conservative
cysteine in the AtVKOR domain, which are directly related to its
oxidoreductase activity.

TABLE 1 | Photosynthetic characterization of wild-type Arabidopsis, vkor mutant lines, and transgenic cysteine-mutant VKORs and VKORWT plants
under normal growth light vs. high light.

517
518
519
520
521
522
523
524
525
526
527
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
551
552
553

Normal growth light (120 mol m2 s1 )

Plants

498
499
500

516

550

496
497

515

528

Levels of D1 Protein Decreased in the

Transgenic Plants with a Conservative
Cysteine VKORs Mutation

493
494

514

2 h High light(600 mol m2 s1 )

554
555

Fv/Fm

PSII

NPQ

Fv/Fm

PSII

NPQ

1.204 0.063 A

0.718 0.008 A

0.436 0.034 A

1.604 0.013 A

557

556

WT

0.865 0.005 A

0.769 0.008 A

501

VKORWT

0.866 0.004 A

0.766 0.013 A

1.201 0.091 A

0.725 0.005 A

0.445 0.008 A

1.612 0.008 A

558

502

VKORC46A

0.861 0.004 A

0.732 0.013 AB

1.151 0.064 A

0.712 0.01 AB

0.421 0.02 AB

1.524 0.036 A

559

503

VKORC230A

0.86 0.002 A

0.729 0.02 AB

1.166 0.061 A

0.702 0.014 AB

0.418 0.017 AB

1.516 0.038 A

560

504

VKORC109A

0.817 0.013 B

0.709 0.02 B

1.026 0.035 B

0.618 0.026 BC

0.353 0.018 B

1.23 0.102 B

561

505

VKORC116A

0.806 0.026 B

0.713 0.046 B

1.012 0.013 B

0.624 0.022 C

0.354 0.022 B

1.225 0.102 B

562

506

VKORC195A

0.699 0.051 B

0.653 0.02 B

0.998 0.011 B

0.605 0.027 DE

0.345 0.016 BC

1.219 0.094 B

563

507

VKORC198A

0.746 0.047 B

0.696 0.037 B

1.008 0.014 B

0.617 0.024 D

0.35 0.011 BC

1.221 0.095 B

564

508

vkor

0.565 0.017 D

0.396 0.071 D

0.905 0.012 C

0.441 0.019 F

0.219 0.017 D

1.043 0.068 C

565

509

VKORC109AC116A

0.662 0.025 C

0.514 0.019 C

0.945 0.01 BC

0.489 0.026 DE

0.278 0.017 C

1.112 0.093 BC

566

510

VKORC195AC198A

0.673 0.025 C

0.525 0.015 C

0.949 0.14 BC

0.493 0.018 E

0.289 0.014 C

1.116 0.089 BC

567

511

The number is reported as the mean SD of three independent measurements for each plant. Significant differences among wild-type Arabidopsis (WT), vkor mutant
lines, and transgenic cysteine-mutant VKORs and VKORWT plants are determined using one-way ANOVA and Ducans Multiple Range Test, indicated with different letters
at P < 0.05 significance level.

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The role of conservative cysteines of AtVKOR in Arabidopsis

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FIGURE 3 | Immunoblot analysis of D1 accumulation in cysteine-mutant VKORs transgenic plants under growth light or in high light. Thylakoid
membrane proteins were extracted from the leaves of AtVKORWT , vkor mutant and cysteine-mutant VKORs plants. The immunoblot was detected by D1-antibody.
Growth light: 120 mol m2 s 1 ; High light: 600 mol m2 s 1 for 2 h.

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to that in AtVKORWT plants (Figures 5 and 6). The results further indicated that the photoprotection mechanism was damaged
in the transgenic plants with mutant AtVKORs of conservative
cysteine.

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591

594
596
597
598
599

604
605
606
607

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647

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Thylakoid protein AtVKOR/LTO1, containing a conserved

VKOR domain with four conservative cysteines, has been
reported to participate in the transmembrane thiol-oxidation
as an oxidoreductase in vitro and in E. coli (Feng et al., 2011;
Karamoko et al., 2011). The necessity of the conservative cysteines of the AtVKOR domain is inferred from the fact that single
or double cysteine mutations lead AtVKOR to losing its function of promoting disulde bond formation in E. coli (Feng et al.,
2011). Whether conservative cysteines of AtVKOR play essential
roles in the complicated plant cells needs to be determined.

595

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643

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Discussion

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642

586

600

639

FIGURE 4 | Quantified analysis of D1 immunoblot. Signals of immunoblot

were quantified using the ImageJ program. The value of D1 accumulation in
AtVKORWT transgenic plants under growth light was adjusted to one. The
relative levels of D1 accumulations in cysteine-mutant VKORs plants
compared to VKORWT plants were, respectively, calculated. The error bars
indicated the SD. Significant differences are determined using one-way
ANOVA and Ducans Multiple Range Test, indicated with different letters at
P < 0.05 significance level. Growth light: 120 mol m2 s 1 ; High light:
600 mol m2 s1 for 2 h.

651
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664

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617
618
619
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621
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623
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625
626
627

667

Accumulation of ROS Increased in the

Transgenic Plants with Conservative
Cysteine VKORs Mutation

668
669
670

In chloroplasts, the damage of PSII assembly is usually associated with the harmful production of ROS, such as H2 O2 ,
O2 , and singlet oxygen (Cejkova et al., 1998; Murata et al.,
2007). Previous studies show that much more H2 O2 and O2
are accumulated in the vkor mutant than in wild-type plants
(Lu et al., 2013). By checking the levels of H2 O2 and O2 in
transgenic plants, we found that the amounts of ROS in plants
with mutations of conservative cysteine VKORs were higher
than that of AtVKORWT plants under growth light (Figures 5
and 6). Under high irradiance, the elevated accumulations of
H2 O2 and O2 in AtVKORC109AC116A and AtVKORC195AC198A
plants were detected, similar to that of the vkor mutant line
(Figures 5 and 6). As to the transgenic plants of AtVKORC46A ,
AtVKORC230A , the levels of H2 O2 and O2 were quite closed

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673
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676
FIGURE 5 | Levels of H2 O2 in cysteine-mutant VKORs transgenic
plants under growth light or in high light. Quantitative accumulations of
H2 O2 were, respectively, detected in 6-weeks-old leaves from WT, vkor
mutant, AtVKORWT, and cysteine-mutant VKORs plants under growth light or
in high light. The error bars indicated the SD. Significant differences are
determined using one-way ANOVA and Ducans Multiple Range Test,
indicated with different letters at P < 0.05 significance level. Growth light:
120 mol m2 s1 ; High light: 600 mol m2 s1 for 2 h.

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The role of conservative cysteines of AtVKOR in Arabidopsis

suggesting the phenotype changes are due to the gene (Lu et al.,
2013). Our investigation further demonstrated that the stunted
growth, the reduced PSII activity, and the aggravated photodamage were related to the damage of AtVKOR oxidoreductase
activity due to the mutation of conservative cysteines in the
AtVKOR domain. Transforming conservative cysteine mutant
VKORs to vkor mutant line, especially double-cysteine mutant
AtVKORs (AtVKORC109AC116A and AtVKORC195AC198A ), could
not eliminate the damage, based on the declined uorescence
parameters (Fv/Fm, PSII and NPQ; Figure 1; Table 1). The high
accumulation of ROS in transgenic plants of AtVKORC109AC116A
and AtVKORC195AC198A further veried the severe photodamage.
The damage of PSII is a primary target of photodamage in
the photosynthetic apparatus (Nishiyama et al., 2001; Murchie
and Niyogi, 2011). To eectively repair the photodamage, diversied photoprotection processes emerge during the evolution of
plants (Kirilovsky and Etienne, 1991; Cai et al., 2010; Lunch
et al., 2013; Nath et al., 2013). The rapid D1 turnover, a cycle
of degradation and re-synthesis of D1 protein, is one of eective
photoprotection methods (Ali et al., 2006; Murata et al., 2007).
The accumulation of D1 protein depends on the balance of the
synthesis and degradation. As to the synthesis of D1 protein,
no dierence has been found at the transcription level of PsbA,
the encoding gene of D1, between vkor decient line and WT
plants (Lu et al., 2013). However, the eects of AtVKOR on the
translation steps of D1 remain to be elucidated. Recent investigations reveal that the de novo synthesis of proteins, particularly
D1 protein, can be inhibited by excess ROS at translation level
(Nishiyama et al., 2001, 2004, 2006, 2011; Murata et al., 2007;
Takahashi et al., 2009). The ROS-induced suppression of protein
synthesis is associated with the specic inactivation of elongation factor G via the formation of an intramolecular disulde
bond (Nishiyama et al., 2006, 2011; Murata et al., 2007). Much
more ROS was accumulated in plants of mutant AtVKORs of
conservative cysteines in our investigation. So it is quite possible that the synthesis of D1 protein in translation steps would
be aected. As to the degradation of D1, when the synthesis of
D1 is blocked, the degradation rate of D1 is accelerated in the
vkor decient line (Yu et al., 2014). The accelerated degradation
of D1 in the vkor line may be related to instability of PSII, since
AtVKOR is required for PSII assembly (Karamoko et al., 2011). In
this investigation, the low accumulation of D1 in the transgenic
plants suggested that the cysteine-dependent activity of AtVKOR
was involved in the D1 turnover-dependent photoprotection
mechanism.
One fast response to high irradiance is to dissipate excessive
thermal energy by NPQ, the core component of which requires
sucient zeaxanthin produced by the xanthophyll cycle (Lunch
et al., 2013). In the xanthophyll cycle, epoxide xanthophyll violaxanthin is rapidly converted via the antheraxanthin to the
de-epoxide zeaxanthin, and zeaxanthin can directly participate in
the dissipation of excess energy (Murchie and Niyogi, 2011). The
key enzyme catalyzing the conversion from antheraxanthin to
zeaxanthin is VDE, a thylakoid luminal protein containing essential disulde bonds (Latowski et al., 2004). Previous investigation
has showed that the xanthophyll cycle in the vkor mutant line is
also impaired under high light, base on the ratio of xanthophyll

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FIGURE 6 | Levels of O2 in cysteine-mutant VKORs transgenic plants

under growth light or in high light. Quantitative accumulation of O2 was,
respectively, detected in 6-weeks-old leaves from WT, vkor mutant,
AtVKORWT, and cysteine-mutant VKORs plants under growth light or in high
light. The error bars indicated the SD. Significant differences are determined
using one-way ANOVA and Ducans Multiple Range Test, indicated with
different letters at P < 0.05 significance level. Growth light: 120 mol m2
s 1 ; High light: 600 mol m 2 s 1 for 2 h.

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In this investigation, our results demonstrated that the

cysteine-dependent oxidoreductase activity of AtVKOR
was directly related to photosynthetic growth in plant. The
AtVKORWT transgenic plants can completely recover the
phenotype defects in the vkor decient line. Similar to the
AtVKORWT , the mutations of non-conservative cysteines in
AtVKOR domain almost rescued the defects of vkor mutant,
though there was a decrease of biomass at about 5% of WT.
Little eects of non-conservative cysteines on the function
of AtVKOR were further shown by checking photosynthetic
parameters, for example Fv/Fm, PSII, and D1 quantity. On
the contrary, the double-mutations of each pair of conservative
cysteines in AtVKOR domain did not compensate the defects
of vkor mutant, displaying a similar phenotype of vkor mutant
lines. The essentiality of the conservative cysteine residues to
activity of VKOR has also been observed in the VKORs from
other species (Li et al., 2010; Wang et al., 2011). Mutations of
each conservative cysteines of MtbVKOR lead mycobacteria
to losing the growing ability in minimal medium (Wang et al.,
2011). The structural analysis shows that four conservative
cysteines are spatially proximate in the active site of VKOR from
Synechococcus sp. (Li et al., 2010). Attentively, it has been proven
that AtVKOR has oxidation, reduction, and isomerization
activity in vitro (Lu et al., 2013). The conservative cysteine of
AtVKOR directly aects electron transferring and is related
to the activity of oxidoreductase (Yang et al., 2015). Based on
all the investigations, it is quite possible that oxidoreductase
activity of AtVKOR aects the thiol-redox metabolism in
chloroplasts and regulates the growth and development of
plants.
AtVKOR is required for the assembly of PSII under growth
light, and the decient vkor mutant line is more susceptible to high light stress, compared with WT (Yu et al., 2014).
Previous study proves that these defects could be abolished
when AtVKORWT is transformed into the vkor mutant line,

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April 2015 | Volume 6 | Article 238

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The role of conservative cysteines of AtVKOR in Arabidopsis

Author Contributions

pigments (Yu et al., 2014). In this investigation, a decline of NPQ

both under growth light and high light was observed in transgenic
plants of VKORC109AC116A and VKORC195AC198A , reecting a
low capability to dissipate excessive irradiance. Presumably, this
is closely related to the declined activity of VDE, whose disulde bonds in active site are not correctly formed due to the
decient oxidoreductase activity of AtVKOR in these transgenic
plants. Altogether, our results suggested that conservative cysteines in AtVKOR domain were related to the oxidoreductase
activity of AtVKOR, and the function was directly involved in
photosynthetic growth and PSII activity in vivo.

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Designed the experiments: X-YW, J-JD. Performed the experiments: J-JD, C-YZ, YL, and H-RC. Analyzed the data: J-JD, C-YZ,
YL, H-RC, and X-YW. Wrote the paper: J-JD and X-YW.

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Acknowledgment

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The present study was supported by the Special Research Fund of

Public Welfare of China Agricultural Ministry (201303093).

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The role of conservative cysteines of AtVKOR in Arabidopsis

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Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or nancial relationships that could be
construed as a potential conict of interest.
Copyright 2015 Du, Zhan, Lu, Cui and Wang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY).
The use, distribution or reproduction in other forums is permitted, provided the
original author(s) or licensor are credited and that the original publication in this
journal is cited, in accordance with accepted academic practice. No use, distribution
or reproduction is permitted which does not comply with these terms.

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Frontiers in Plant Science | www.frontiersin.org

April 2015 | Volume 6 | Article 238