The Plant Cell, Vol. 19: 3157–3169, October 2007, www.plantcell.org ª 2007 American Society of Plant Biologists
Arabidopsis Cotyledon-Specific Chloroplast Biogenesis Factor
CYO1 Is a Protein Disulfide Isomerase
W
Hiroshi Shimada,a,1,2 Mariko Mochizuki,a,1 Kan Ogura,a John E. Froehlich,b Katherine W. Osteryoung,c
Yumiko Shirano,d Daisuke Shibata,e Shinji Masuda,a Kazuki Mori,a and Ken-ichiro Takamiyaa
a Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama
226-8501, Japan
b Michigan State University–Department of Energy Plant Research Laboratory, Michigan State University, East Lansing,
Michigan 48824
c Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824
d Boyce Thompson Institute for Plant Research, Ithaca, New York 14853
e Kazusa DNA Research Institute, Kisarazu, Chiba 292-0812, Japan
Chloroplast development in cotyledons differs in a number of ways from that in true leaves, but the cotyledon-specific program
of chloroplast biogenesis has not been clarified. The cyo1 mutant in Arabidopsis thaliana has albino cotyledons but normal
green true leaves. Chloroplasts develop abnormally in cyo1 mutant plants grown in the light, but etioplasts are normal in
mutants grown in the dark. We isolated CYO1 by T-DNA tagging and verified that the mutant allele was responsible for the
albino cotyledon phenotype by complementation. CYO1 has a C4-type zinc finger domain similar to that of Escherichia coli
DnaJ. CYO1 is expressed mainly in young plants under light conditions, and the CYO1 protein localizes to the thylakoid
membrane in chloroplasts. Transcription of nuclear photosynthetic genes is generally unaffected by the cyo1 mutation, but the
level of photosynthetic proteins is decreased in cyo1 mutants. Recombinant CYO1 accelerates disulfide bond reduction in the
model substrate insulin and renatures RNase A, indicating that CYO1 has protein disulfide isomerase activity. These results
suggest that CYO1 has a chaperone-like activity required for thylakoid biogenesis in cotyledons.
INTRODUCTION
In dicotyledonous plants, the development of cotyledons—
embryonic leaves formed during embryogenesis—is distinct
from that of true leaves, which arise as the result of apical meristem activity. Although both cotyledons and true leaves contain
chloroplasts and are photosynthetically active, chloroplast differentiation follows distinct paths in these two organs (Mansfield
and Briarty, 1996). In cotyledons, plastids partially develop during embryogenesis, but development stops during seed maturation and dormancy. Upon germination in light, the plastids
further develop into functional chloroplasts. By contrast, during
true leaf development, proplastids differentiate into mature
chloroplasts. Fully differentiated chloroplasts in cotyledons resemble young true leaf chloroplasts, although they usually contain a less extensive thylakoid membrane than mature true leaf
chloroplasts (Deng and Gruissem, 1987).
As long as seedlings grow submerged in the soil, they live
heterotrophically, using nutrients stored mainly in cotyledon cells.
During this initial stage of development, etioplasts develop from
1 These
authors contributed equally to this work.
correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Hiroshi Shimada
([email protected]).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.107.051714
2 Address
proplastids. Once the seedling emerges from the soil and reaches
light, it rapidly transforms etioplasts into chloroplasts, thereby
enabling photosynthesis and switching from heterotrophic to
autotrophic growth. In contrast with cotyledons, true leaf chloroplasts develop directly from proplastids. Mutants with pigment
deficiencies that are confined either to cotyledons or true leaves
also suggest differences in the regulation of plastid development
in these two organs. Arabidopsis thaliana plants with mutations in
VAR1 and VAR2 (FtsH proteases) have normal green cotyledons
(Sakamoto et al., 2003), whereas a number of other Arabidopsis
mutants, including wco (unidentified gene product) (Yamamoto
et al., 2000), sco1 (chloroplast elongation factor G) (Albrecht
et al., 2006), and sig2 and sig6 (sigma factors) (Privat et al., 2003;
Ishizaki et al., 2005), have albino or pale-green cotyledons but
normal green leaves. Despite these reports, how cotyledonspecific chloroplasts develop remains largely unknown.
In Escherichia coli, DnaJ, a primary Hsp40 homolog, acts in
conjunction with the HSP70 protein DnaK and the nucleotide
exchange factor GrpE during many cellular processes, including protein folding, protein transport, degradation of misfolded
proteins, and bacteriophage DNA replication (for reviews, see
Georgopoulos, 1992; Cyr et al., 1994). DnaJ has chaperone
activity, as revealed by its capacity to recognize nonnative
proteins and prevent the aggregation of folding intermediates
in vitro (Langer et al., 1992; Szabo et al., 1996; Goffin and
Georgopoulos, 1998). DnaJ can also catalyze the formation,
reduction, and isomerization of disulfide bonds similar to protein
disulfide isomerase (PDI) (de Crouy-Chanel et al., 1995). PDI is a
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Figure 1. The cyo1 Mutant Plant Has Albino Cotyledons.
A 10-d-old wild-type plant (A), a cyo1 mutant plant (B), and a cyo1 mutant plant complemented with the CYO1 genomic DNA clone (C). All were grown
under continuous light at 238C on 1/2 MS agar plate medium with 1.5% sucrose.
molecular chaperone that catalyzes dithiol/disulfide interchange
reactions and promotes protein disulfide formation, isomerization, or reduction, depending on the redox potential and nature of
the polypeptide substrate (Holmgren, 1979; Freedman, 1989;
Gilbert, 1990). Zn2, one of two C4-type zinc finger motifs in DnaJ,
is important for this enzymatic activity (Goffin and Georgopoulos,
1998; Tang and Wang, 2001; Shi et al., 2005).
In plants, DnaJ-like proteins are involved in plastid biogenesis.
Or is involved in the differentiation of noncolored plastids into
chromoplasts for carotenoid accumulation (Lu et al., 2006).
BSD2 is required for posttranslational regulation of the L subunit
of ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco)
(Brutnell et al., 1999). Chloroplast DnaJ homolog2 (CDJ2) is
required for the biogenesis and/or maintenance of thylakoid
membranes (Liu et al., 2005). RB60 is an atypical PDI that
functions as a member of a redox regulatory protein complex
controlling translation in Chlamydomonas reinhardtii chloroplasts (Kim and Mayfield, 1997).
We now describe an Arabidopsis mutant, cyo1, with albino
cotyledons and cotyledon-specific defects in chloroplast biogenesis. The CYO1 protein has a Zn2 motif similar to that in E. coli
DnaJ and has thiol disulfide reduction activity. We also demonstrate that CYO1 is a thylakoid membrane protein and that CYO1
is expressed mainly in cotyledons under illumination. These
results suggest a role for CYO1 specifically in cotyledon chloroplast differentiation.
flowering was similar in cyo1 mutants and wild-type plants. No
chlorophyll could be detected in cotyledons from mutants grown
in normal (26 mmolm2s1) or very dim (0.70 mmolm2s1) light
conditions. These results suggest that the albino cotyledons
observed in the mutants were not the result of photobleaching or
photoinhibition. The chlorophyll content in mutant rosette leaves
was comparable to that in wild-type leaves (see Supplemental
Figure 1 online).
Ultrastructure of Plastids in cyo1 Mutants
To further characterize the cyo1 mutant, we examined plastid
ultrastructure by transmission electron microscopy. In cotyledons
RESULTS
Characterization of the cyo1 Mutant
T-DNA insertional mutants in Arabidopsis thaliana were produced on a large scale by vacuum infiltration to identify mutants
with chloroplast development phenotypes (Shirano et al., 2000).
Several mutants with an albino cotyledon phenotype were
isolated from the ;3,500 transgenic lines examined. One such
mutant, designated cyo1 (shi-yo-u means cotyledon in Japanese), had albino cotyledons but normal green leaves (Figure
1B). On 1/2 MS (for half-concentration of normal Murashige and
Skoog) plates containing 1.5% sucrose, germination of cyo1
mutant seeds was normal but expansion of the first leaf was
slower than in the wild type. In soil or on 1/2 MS plates containing
no sucrose, many mutants could not produce leaves and subsequently died. Mutants with leaves that were transferred from
plates to soil grew autotrophically and produced mature seeds
by self-pollination. The period between first leaf expansion and
Figure 2. The Chloroplasts in Cotyledons of cyo1 Mutants Are Small and
Abnormal in Shape.
Wild-type chloroplast (A) and cyo1 mutant plastid (B) in cotyledons from
7-d-old plants grown under light. The magnification is the same in (A) and
(B). Wild-type (C) and cyo1 mutant (D) etioplast in cotyledons from 7-dold plants grown in the dark. The magnification is the same in (C) and (D).
Arabidopsis Protein Disulfide Isomerase CYO1
from wild-type plants grown under light conditions, chloroplasts
were crescent-shaped and contained thylakoid membranes,
including stroma thylakoids and grana thylakoids (Figure 2A). In
albino cotyledons of cyo1 mutant plants, the plastids were
smaller and abnormally shaped (Figure 2B). The plastids did
not develop thylakoid membranes but instead contained a large
number of electron-dense particles (Figure 2B). Leaf chloroplasts from cyo1 mutant and wild-type plants were similar ultrastructurally (see Supplemental Figure 2 online), as were etioplasts
in cotyledons from wild-type and mutant plants grown in the dark
(Figures 2C and 2D). These results indicate that the cyo1 mutation affected chloroplast biogenesis under light conditions but
did not affect etioplast biogenesis under dark conditions.
Identification and Characterization of the CYO1 Gene
cyo1 plants were reciprocally backcrossed to wild-type plants,
and segregation of the albino cotyledon phenotype was examined in the F1 and F2 progeny. All of the F1 progeny showed wildtype cotyledons (green color) and hygromycin resistance. In F2
progeny, the hygromycin sensitivity segregated ;3:1 (hygromycinresistant ¼ 177, hygromycin-sensitive ¼ 54; x2 ¼ 0.32, P > 0.05)
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and the cotyledon phenotype segregated ;2:1 (hygromycinresistant/green cotyledon ¼ 129, hygromycin-resistant/albino
cotyledon ¼ 48; x2 ¼ 3.08, P > 0.05). These results indicate that
the mutant phenotype segregated as a single recessive mutation
and cosegregated with the T-DNA, suggesting that the mutation
was caused by insertion of the T-DNA into the nuclear genome.
DNA gel blot hybridization analysis indicated that the cyo1
mutant genomic DNA contained a single T-DNA (see Supplemental Figure 3 online).
To determine the T-DNA insertion site, we isolated the T-DNA
by plasmid rescue and sequenced the flanking DNA. The T-DNA
was inserted in exon 1 of At3g19220, which had been annotated
previously (accession number AY059925) as an unknown proteincoding gene. Because the T-DNA contained a set of concatenated enhancer fragments derived from the 35S promoter of
Cauliflower mosaic virus for activation tagging, it was possible
that the cyo1 mutant phenotype could be due either to transcriptional activation of mutant CYO1 or neighboring genes or to
disruption of CYO1. However, heterozygous cyo1/CYO1 plants
exhibited normal green cotyledons, arguing against the former
possibility. In addition, the albino cotyledon phenotype in cyo1
homozygotes was rescued by introduction of a wild-type
Figure 3. Molecular Characterization of the CYO1 Gene.
(A) Structure of CYO1. Boxes and bars represent exons and introns, respectively.
(B) Structure of the CYO1 cDNA. The black box and white boxes represent the open reading frame and untranslated region, respectively.
(C) to (E) DNA fragments used for the complementation assay. The DNA fragment in (C) contains the ATG codon at 3 bp, whereas the DNA fragments
between 5 and 606 bp, shown in (D) and (E), do not. The ATG at 32 bp was mutated to ATT in the DNA fragment shown in (E). 35S represents the
cauliflower mosaic virus 35S promoter.
Figure 4. Homologs and Putative Topology of CYO1.
(A) Alignment of the amino acid sequence of CYO1 (accession number AY059925) and CYO1 homologs from potato (TC133656), tomato (TC180110),
Medicago truncatula (ABE80527), soybean (TC228649), poplar (TC44537), pine (TC76401), spruce (TC24344), maize (TC343209), rice (TC332052), and
wheat (TC257683). Sequences of potato, tomato, soybean, poplar, pine, spruce, maize, rice, and wheat were drawn from The Institute for Genomic
Research Gene Indices (http://tigrblast.tigr.org/tgi/). The sequences of spruce, maize, and wheat are not full-length amino acid sequences. Conserved
residues are shown in black, and similar residues are shown in gray. Asterisks indicate Cys residues, the bar shows the zinc finger motif, and the vertical
arrow indicates the putative cleavage site of the transit peptide of Arabidopsis CYO1.
(B) Putative topology of the zinc finger motif in CYO1. Residues conserved between CYO1 and DnaJ are highlighted. Residues conserved in the zinc
finger domain are shown in black, and identical residues between CYO1 and DnaJ are shown in gray.
(C) The topology of E. coli DnaJ (Shi et al., 2005).
Arabidopsis Protein Disulfide Isomerase CYO1
At3g19220 genomic fragment (Figure 1C). These results confirm
that At3g19220 is CYO1.
The sequence of a full-length At3g19220 cDNA was reported
previously by Seki et al. (2002). We cloned the full-length cDNA
by RT-PCR and confirmed the sequence of the amplified fragment. The 959-bp At3g19220 cDNA has three exons (Figure 3A).
The T-DNA insertion site was in the first exon, at 19 bp. The cDNA
had putative translation initiation codons (ATG) at 3 and 32 bp. To
analyze which of the two possible initiation codons is used, cDNA
fragments between þ1 and þ606 bp, or between þ5 and þ606
bp, were ligated into a vector under the control of the cauliflower
mosaic virus 35S promoter, and the vector was introduced into
the cyo1 mutant. If the ATG at 3 bp was the initiation codon for
translation, the stop codon would have to be at 294 bp (TAG),
whereas if the ATG at 32 bp was the initiation codon, the stop
codon would have to be at 595 bp (TGA). Only the vector containing the cDNA between þ5 and þ606 bp could complement
the cyo1 phenotype (Figures 3C and 3D). Furthermore, to exclude the possibility that At3g19220 encoded a noncoding RNA,
we constructed a vector containing the cDNA between 5 and 606
bp with the ATG at 32 bp mutated to ATT. When this construct
was introduced into mutant plants, transformants harboring the
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vector had albino cotyledons (see Supplemental Figure 4 online).
These results indicate that the translation initiation codon of
At3g19220 is at 32 bp.
At3g19220/CYO1 encodes a protein of 187 amino acids with a
putative molecular weight of 20,842. CYO1 contains nine Cys
residues and has a predicted zinc finger–like domain between
residues 135 and 164 similar to that found in DnaJ (Figures 4B
and 4C). A BLAST search revealed that many plants (e.g., potato
[Solanum tuberosum], tomato [Solanum lycopersicum], Medicago
truncatula, soybean [Glycine max], poplar [Populus sp], pine [Pinus
sp], spruce [Picea abies], maize [Zea mays], rice [Oryza sativa], and
wheat [Triticum aestivum]) have a CYO1 homolog (Figure 4A).
CYO1 homologs could not be identified in moss or algae.
Expression Analysis of CYO1
We analyzed CYO1 mRNA levels in 7-d-old wild-type and cyo1
mutant plants. The CYO1 transcript was undetectable by RNA
gel blot hybridization; thus, nonsaturating RT-PCR conditions
were used for quantification. As shown in Figure 5A, the wildtype transcript but not the cyo1 mutant transcript could be
Figure 5. RNA Expression and Immunoblot Analysis of CYO1.
(A) Analysis of CYO1 transcripts from 7-d-old wild-type (lane 1) and cyo1 mutant (lane 2) plants grown under light. Ubiquitin10 (UBQ10) was analyzed as
a loading control.
(B) Immunoblot analysis of CYO1 levels in 7-d-old wild-type (left lane) and cyo1 mutant (right lane) plants grown under light conditions. Fifty micrograms
of total protein was loaded in each lane. The arrow indicates CYO1. Plasma Aquaporin (PAQ) was analyzed as a loading control.
(C) Effect of light on CYO1 transcript levels. Seven-day-old wild-type Arabidopsis plants grown in continuous dark were illuminated for the times
indicated (0 to 12 h; lanes 1 to 6). Lane 7 shows 7-d-old plants grown under continuous light.
(D) Effect of light on CYO1 protein levels. Seven-day-old wild-type Arabidopsis plants grown in continuous dark were illuminated for the times indicated
(0 to 12 h; lanes 1 to 4). Lane 5 shows 7-d-old plants grown under continuous light. Fifty micrograms of total protein was loaded in each lane.
(E) Organ-specific transcripts of CYO1 in continuously illuminated adult plants. Total RNA was isolated from 4-week-old wild-type Arabidopsis for
organ-specific quantification of the CYO1 transcript (lanes 2 to 5).
(F) Organ-specific CYO1 protein levels. Fifty micrograms of total protein was loaded in each lane.
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detected. To analyze the amount of CYO1 protein in plants, we
prepared a rabbit antibody against recombinant CYO1 and
performed protein gel blotting. Total protein from 7-d-old Arabidopsis grown under light on 1/2 MS plates containing 1.5%
sucrose was separated by SDS-PAGE. Anti-CYO1 recognized a
band with a molecular weight of ;20,000 in the wild type,
consistent with the predicted molecular weight, but not in cyo1
mutant samples (Figure 5B). These results indicate that the
abnormal chloroplast phenotype observed in cyo1 mutant plants
was caused by a lack of CYO1 protein.
The CYO1 transcript level was very low in etiolated seedlings,
but upon illumination it gradually increased and reached a maximum at 6 h (Figure 5C). Similarly, CYO1 protein was not detected
in etiolated seedlings but increased upon illumination (Figure 5D).
Although CYO1 transcript levels in seedlings illuminated for 6 h
were almost the same as in 7-d-old seedlings grown under
continuous light, CYO1 protein levels in seedlings illuminated for
12 h did not reach those of 7-d-old seedlings grown under
continuous light.
Wild-type plants grown for 7 d in the light expressed CYO1, but
significantly less CYO1 expression was detected in the organs of
4-week-old Arabidopsis (Figures 5E and 5F). These results
indicate that CYO1 is expressed mainly in young plants grown
under light conditions.
Subcellular Localization of CYO1
The TargetP program (http://www.cbs.dtu.dk/services/TargetP/)
predicted a mitochondrial presequence between residues 1 and
Figure 6. Localization of CYO1 Protein.
(A) In vitro chloroplast import and protease protection assay for CYO1.
35S-labeled CYO1 and RBC-S were produced by in vitro translation. The
proteins were incubated with isolated pea chloroplasts in the presence of
4.0 mM Mg-ATP for 30 min at room temperature. Chloroplasts were
recovered by sedimentation through 40% (v/v) Percoll. The recovered
intact chloroplasts were then incubated without (lanes 2, 3, 7, and 8) or
with (lanes 4, 5, 9, and 10) thermolysin for 30 min at 48C. Intact
chloroplasts were recovered by centrifugation through 40% (v/v) Percoll
and fractionated into total membrane (Pellet; lanes 2, 4, 7, and 9) and
soluble (Supernatant [Sup.]; lanes 3, 5, 8, and 10) fractions. CYO1 was
detected in the chloroplast fraction (Pellet), as was the control protein. TP
represents 10% of in vitro translated products (lanes 1 and 6).
(B) Immunoblot analysis of CYO1. Total protein is whole cell protein (lane
1). Intact chloroplasts (lane 2) were lysed, and soluble (Sup.; lane 3) and
insoluble (Pellet; lane 4) proteins (10 mg/lane) were separated by 12.5%
SDS-PAGE. Each protein fraction was prepared from 7-d-old plants.
RBC-L (stroma protein), E37 (chloroplast envelope protein), PAQ (plasma
membrane protein), and BiP (ER protein) were analyzed as controls for
each fraction.
(C) Sonicated thylakoids were prepared from 7-d-old plants and were
treated with 0.1 M sodium carbonate (lanes 2 and 3), 1.0% Nonidet P-40
(NP40; lanes 4 and 5), or 2.0 M NaCl (lanes 6 and 7) and then soluble
(Sup.; lanes 3, 5 and 7) and insoluble (Pellet; lanes 2, 4, and 6) fractions
were separated at 12,000g. LHCP and FNR were used as marker
proteins for intrinsic and extrinsic thylakoid membrane proteins, respectively.
(D) Sonicated thylakoids were prepared from 7-d-old plants and were
treated with thermolysin (0, 10, or 100 mg/mL) for 60 min on ice. FNR and
PSI-N were used as stroma- and lumen-exposed peripheral thylakoid
protein markers, respectively.
(E) Intact thylakoids of 7-d-old plants were solubilized by treatment with
1% n-dodecyl-b-D-maltoside and separated by 5 to 14% blue native gel
electrophoresis. After electrophoresis, the gel was incubated in 0.1%
SDS containing transfer buffer (100 mM Tris, 192 mM Gly, and 5%
methanol) for 10 min at room temperature and blotted to a membrane
(left panel). The membrane was reacted with antibodies against CYO1
(right panel). Without additional staining, protein complexes were detected by bound Coomassie blue dye and chlorophylls. Bands corresponding to various photosynthetic complexes are indicated (Asakura
et al., 2004) (see also http://www.hos.ufl.edu/clineweb/BNgel.htm). Ferritin (880 and 440 kD) and BSA (132 and 66 kD) were used as molecular
mass marker proteins. Arrowheads show the bands of CYO1. Closed
arrowheads show that CYO1 bands comigrate with PSI/LHCI and PSII/
LHCII, and the open arrowhead shows the CYO1 band in an unidentified
thylakoid protein complex.
Arabidopsis Protein Disulfide Isomerase CYO1
30 in CYO1, but the WoLF PSORT program (http://wolfpsort.
seq.cbrc.jp/) (Horton et al., 2007) suggested that CYO1 is a
chloroplast protein. The potato, tomato, Medicago truncatula,
soybean, poplar, pine, and rice proteins were also predicted to
be chloroplastic by TargetP. To investigate CYO1 localization,
we performed an in vitro chloroplast import experiment. Fulllength 35S-labeled CYO1 was produced by in vitro translation. A
control assay was performed with the stroma-localized small
subunit of Rubisco (RBC-S). After incubation of the CYO1 translation product with isolated pea (Pisum sativum) chloroplasts,
radiolabeled CYO1 was detected in the chloroplast fraction, as
was control protein (Figure 6A). When the chloroplasts were
treated with thermolysin, a protease that does not penetrate the
envelope membrane, mature CYO1 and RBC-S were protected
from proteolysis (Figure 6A). These results indicate that CYO1 is
targeted to chloroplasts. Although the calculated molecular
weight of full-length CYO1 is 20,842, the apparent molecular
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weight of the full-length CYO1 translation product was ;25,000
by SDS-PAGE. After incubation of this CYO1 translation product
with isolated pea chloroplasts, a protein with a molecular mass of
;20,000 was detected. These results suggest that CYO1 contains a chloroplast transit peptide.
To further determine the subcellular distribution of CYO1,
Arabidopsis chloroplasts were isolated by two-step Percoll gradient centrifugation (Aronsson and Jarvis, 2002) and lysed by
osmotic disruption. Soluble and insoluble fractions separated by
a low-speed centrifugation (3000g, 5 min) that sediments the
thylakoid but not the envelope membranes were analyzed by
protein gel blotting using the anti-CYO1 antibody. CYO1 was detected in the chloroplast pellet fraction (Figure 6B), which contained the thylakoid membrane marker protein LHCP but not the
stroma protein large subunit of Rubisco (RBC-L), the envelope
protein E37, a major envelope protein (Teyssier et al., 1996), the
plasma membrane protein aquaporin PAQ (Ohshima et al., 2001),
Figure 7. RNA Expression and Immunoblot Analysis of Chloroplast Proteins.
(A) Total RNA was prepared from 7-d-old wild-type (lane 1) and cyo1 mutant (lane 2) plants. One microgram of total RNA was loaded, and transcript
levels were measured in wild-type (odd-numbered lanes) and cyo1 mutant (even-numbered lanes) plants for the following nuclear genes: RBC-S, LHCP,
and 18S (18S rRNA); the following chloroplast genes transcribed by PEP: RBC-L, PSB-A (D1), PSB-B (CP47), and PSA-A (PsaA); and the following
chloroplast genes transcribed by NEP: ACCD and RPO-B.
(B) Twenty micrograms of total protein of 7-d-old wild-type (lane 1) and cyo1 mutant (lane 2) plants was loaded. The SDS-PAGE gel (20 mg of protein per
lane) was stained with Coomassie blue. The arrowheads show the major bands of RBC-L, RBC-S, and LHCP.
(C) Immunoblot analysis of photosynthesis and control proteins (20 mg of protein per lane) in 7-d-old wild-type (odd-numbered lanes) and cyo1 mutant
(even-numbered lanes) plants. D1, D2, and CP43 are proteins of PSII, Cyt f is a protein of cytochrome b6f, PSI-AB and PSI-N are proteins of PSI, FNR is
ferredoxin NADPþ oxidoreductase, TRX-M is thioredoxin m, RBC-L is the large subunit of Rubisco, CF1-b and CF1-g are subunits of chloroplast
ATPase, E37 is a chloroplast envelope protein, ACCD is acetyl-CoA carboxylase, and BiP is an ER protein.
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or the endoplasmic reticulum (ER) protein BiP (Hatano et al., 1997).
This result indicates that CYO1 is a thylakoid membrane protein.
To confirm that CYO1 was a thylakoid membrane protein, we
treated sonicated thylakoid fractions with 0.1 M sodium carbonate, 1.0% Nonidet P-40, or 2.0 M sodium chloride (Figure 6C).
Neither CYO1 nor LHCP was extracted by sodium carbonate or
sodium chloride, but both were solubilized by Nonidet P-40. By
contrast, FNR (for ferredoxin NADPþ oxidoreductase), which is
attached to the thylakoid membrane, was partially extracted by
sodium carbonate and sodium chloride (Figure 6C).
To analyze the topology of CYO1 in the thylakoid membrane,
we performed a protease protection assay (Motohashi and
Hisabori, 2006). Incubation of sonicated thylakoid membrane
samples with the protease thermolysin resulted in the degradation of FNR, which is located on the stroma-exposed surface of
thylakoid membranes, and PSI-N (for the N subunit of photosystem I), which is located on the lumen-exposed surface of the
thylakoid membrane. By contrast, CYO1 was not degraded
(Figure 6D). These results suggest that CYO1 is an intrinsic
thylakoid membrane protein. Since CYO1 contains no predicted
membrane-spanning segments, it may be protected from thermolysin degradation by its association with other thylakoid
membrane proteins.
To begin investigating whether CYO1 might be in a complex
with other thylakoid proteins, we performed blue native gel
electrophoresis (Asakura et al., 2004) (Figure 6E, left) and immunoblotting (Figure 6E, right) with thylakoids isolated from 7-d-old
wild-type Arabidopsis plants and solubilized with 1% n-dodecylb-D-maltoside. CYO1 comigrated with the PSI/LHCI and PSII/
LHCII complexes (closed arrowheads, 1030 and 710 kD). It was
also detected in a band of 220 kD that migrated between
cytochrome b6f and the LHCII trimer (open arrowhead). These
results suggest that CYO1 associates with multiple thylakoid
complexes in vivo.
Expression of Photosynthetic Genes and Proteins
Figure 8. CYO1 Has Reductase and Oxidase Activity.
(A) Purified D30CYO1 catalyzes the reduction of insulin. The reaction was
initiated by adding DTT into 0.1 M potassium phosphate, pH 6.6,
containing 0.13 mM bovine insulin in the absence (circles) or presence
of 1.0 mM D30CYO1 (diamonds) or 1.0 mM DnaJ (squares). The reduction
of insulin and the resulting precipitation of the B chain was monitored by
following the optical density at 650 nm. Data represent means 6 SD of
three independent experiments.
(B) Effect of CYO1 on refolding of reduced and denatured RNase A.
Refolding of denatured and reduced RNase A (40 mM) was initiated by
10-fold dilution in 50 mM Tris-HCl, pH 8.0, containing 0.1 M NaCl and 0.3
mM DTT at 308C in the absence (circles) or presence of 1.0 mM D30CYO1
(diamonds) or 1.0 mM DnaJ (squares). At the indicated time points, an
aliquot containing 40 mmol of RNase A was withdrawn from the reaction
to assay RNase A activity. Activity is expressed as a percentage of native
RNase A activity and represents the average 6 SD of three independent
experiments.
(C) Effect of metal on CYO1 activity. Purified D30CYO1 was denatured
with 6.0 M guanidine hydrochloride, and then the proteins were renatured with dialyzing buffer containing 5.0 mM ZnCl2, CuCl2, CaCl2,
We analyzed transcript levels for several chloroplast and nuclear
photosynthetic genes in 7-d-old wild-type and cyo1 mutant
plants (Figure 7A). The nuclear transcripts we tested encoded
RBC-S and LHCP, and the chloroplast transcripts tested encoded the RBC-L, the A1 protein of PSI (PSA-A), the D1 protein of
PSII (PSB-A), the CP47 protein of PSII (PSB-B), acetyl-CoA
carboxylase (ACCD), and the b-subunit of plastid-encoded RNA
polymerase (RPO-B). RBC-L, PSA-A, PSB-A, and PSB-B are
transcribed by plastid-encoded RNA polymerase (PEP), whereas
ACCD and RPO-B are transcribed by nucleus-encoded RNA
polymerase (NEP) (Hess and Borner, 1999; Kanamaru and
Tanaka, 2004). Transcript levels of all genes tested were comparable in wild-types and cyo1 plants, with the exception of
MgCl2, or no divalent metal ion. The dialyzed proteins were centrifuged,
and the supernatant was used for enzymatic assays. The reduction of
insulin and the resulting precipitation of the B chain were monitored by
following the optical density at 650 nm. Data represent means 6 SD of
three independent experiments.
Arabidopsis Protein Disulfide Isomerase CYO1
ACCD and RPO-B, which were significantly increased in the
mutant. These results suggest that NEP-mediated gene transcription was increased by the cyo1 mutation, whereas PEPmediated transcription was not influenced by the mutation.
To analyze the relative amounts of chloroplast proteins in the
mutant and the wild type, we performed SDS-PAGE and protein
gel blotting. Coomassie blue staining revealed that the density of
the three major bands, LHCP, RBC-L, and RBC-S, was less in
cyo1 mutant plants than in wild-type plants (Figure 7B, lanes
1 and 2). For protein gel blotting, we used antibodies against seven
nucleus-encoded proteins—PSI-N, FNR, TRX-M (thioredoxin m),
LHCP, CF1-g (chloroplast ATPase), E37 (envelope protein), and
BiP (ER protein)—and eight chloroplast-encoded proteins—D1,
D2, and CP43 (PSII), cytochrome f (cytochrome b6f), PSI-AB (PSI
protein), RBC-L, CF1-b (chloroplast ATPase), and ACCD. The
levels of D1, D2, CP43, cytochrome f, PSI-AB, PSI-N, LHCP,
RBC-L, CF1-b, and CF1-g were significantly lower in cyo1 mutants than in the wild type (Figure 7C). The levels of FNR, TRX-M,
E37, and BiP were comparable in wild-type and cyo1 plants, and
the level of ACCD was increased significantly in the mutant.
CYO1 Has Protein Zn-Dependent Disulfide
Isomerase Activity
CYO1 residues 135 to 165 are similar to the C4-type zinc finger-2
(Zn2) of E. coli DnaJ (Figures 4B and 4C). Zn2 is important for the
enzymatic activity of DnaJ (Tang and Wang, 2001), such as
reduction of insulin disulfides and protein disulfide formation
(e.g., oxidative renaturation of reduced RNase) (de Crouy-Chanel
et al., 1995). To analyze the catalytic properties of CYO1, we tried
to express full-length CYO1 in E. coli, but it was insoluble under
all E. coli growth/detergent conditions we tried. Therefore, we
expressed a truncated CYO1 that included residues 31 to 187
(D30CYO1) (data not shown). This D30CYO1 was soluble and
could be used for rapid spectrophotometric assay of disulfide
isomerase activity based on the reduction of insulin (Holmgren,
1979). Mature insulin contains two polypeptide chains, A and B,
linked by disulfide bonds. When these bonds are broken, the free
B chain is insoluble and precipitates, increasing absorbance at
650 nm. DnaJ in E. coli catalyzes the DTT-dependent reduction
of insulin (de Crouy-Chanel et al., 1995; Tang and Wang, 2001;
Shi et al., 2005). We measured the reduction of insulin (130 mM)
by DTT (0.3 mM) in the presence of 1.0 mM D30CYO1 or DnaJ
(Figure 8A). Insulin B chain precipitation was observed after 30
min in the presence of D30CYO1 or DnaJ and leveled off after 60
min. Significantly less B chain precipitated in the absence of
D30CYO1 or DnaJ.
Renaturation of reduced, denatured RNase A, which contains
eight sulfhydryl groups, involves the oxidation of its thiol groups
followed by rearrangement of the disulfides to the native conformation (with four disulfide bridges) (Anfinsen and Scheraga,
1975). As reported previously (Pigiet and Schuster, 1986), the
spontaneous refolding of reduced, denatured RNase A was
negligible in air (Figure 8B). Addition of D30CYO1 or DnaJ to the
reaction stimulated RNase A renaturation. The renaturation
activity of D30CYO1 was lower than that of DnaJ; however, it
may be that the reduced, denatured RNase A is a better substrate for DnaJ than for D30CYO1. Nevertheless, D30CYO1 cat-
3165
alyzed the reactivation of reduced, denatured RNase A,
indicating that CYO1 has PDI activity.
To determine whether CYO1 requires Zn2þ for its enzymatic
activity, as does DnaJ, purified D30CYO1 was denatured and
renatured in a dialyzing buffer containing ZnCl2, CuCl2, CaCl2,
MgCl2, or no divalent metal ion. D30CYO1 catalyzed the reduction of insulin when renatured in the presence of ZnCl2 but lacked
activity when renatured in the presence of any of the other buffers
(Figure 8C). This result indicates that Zn2þ is required for the
enzymatic activity of CYO1.
DISCUSSION
In this study, we selected a cotyledon-specific albino mutant of
Arabidopsis and isolated the gene CYO1, which encodes a
thylakoid membrane protein with a DnaJ zinc finger domain.
CYO1 copurifies with the PSI/LHCI and PSII/LHCII complexes
and has PDI activity.
Many dicotyledonous and monocotyledonous plants have
CYO1 homologs. The C-terminal region is highly conserved
among CYO1 homologs, suggesting that this region is critical for
CYO1 function. Furthermore, Arabidopsis CYO1 has nine Cys
residues, all of which are conserved in potato and tomato. Seven
of these Cys residues are also conserved in all plants (Figure 4A).
Although the phenotype of monocotyledonous plants carrying
the cyo1 mutation awaits future investigation, the conservation of
Cys residues suggests that they are important for CYO1 function
in all angiosperms.
CYO1 transcript and protein accumulate in seedlings and are
increased by light irradiation (Figures 5C and 5D). In cyo1
mutants grown under light conditions, the plastids in cotyledons
are immature and do not develop any thylakoid membrane
(Figure 2B). However, in cyo1 mutants grown under continuous
dark, the etioplasts in cotyledons are comparable to those in
Table 1. Number of Cys Residues in Subunits of PSI and PSII
in Arabidopsis
Protein
PSI
Hydrophobic subunits
PsaA (A1)
PsaB (A2)
PsaG
PsaI
PsaJ
PsaK
Hydrophilic subunits
Stromal orientation
PsaC
PsaD
PsaE
PsaH
Lumenal orientation
PsaF
PsaN
Number
of Cys
Residues Protein
4
2
0
0
0
2
9
1
0
0
3
5
PSII
Hydrophobic subunits
D1
D2
CP47
CP43
Cytochrome b559
a Subunit
b Subunit
PsbH
PsbN
22 kD
Hydrophilic subunits
33 kD
23 kD
17 kD
10 kD
Number
of Cys
Residues
2
4
3
4
0
0
0
0
0
4
1
2
0
3166
The Plant Cell
wild-type plants. Seed maturation in cyo1 mutants is normal;
thus, CYO1 likely functions only during chloroplast development
in cotyledons.
The cyo1 mutation influences the accumulation of many chloroplast proteins, but it does not affect the accumulation of
nuclear transcripts. To analyze the effect of the cyo1 mutation on
transcription, we performed a DNA array assay for cyo1 mutant
and wild-type plants (see Supplemental Figure 5 online). Scatterplots of mRNA accumulation for 2880 genes differed very little
between wild-type and cyo1 plants, suggesting that cyo1 does
not influence the transcription of nuclear genes. However, transcript levels for some chloroplast genes (ACCD and RPO-B)
transcribed by NEP were increased in the mutant. NEP is largely
responsible for the transcription of housekeeping genes during
early chloroplast development (Hanaoka et al., 2005); therefore,
only chloroplast genes transcribed by NEP are likely to be
influenced by the cyo1 mutation.
CYO1 residues 135 to 165 are similar to the Zn2 domain of E.
coli DnaJ. DnaJ proteins are molecular chaperones that specifically regulate DnaK-like proteins involved in protein folding.
Hsp70s, DnaK homologs in chloroplasts, play an important role
in a number of processes (Liu et al., 2005; Lu et al., 2006). However, CYO1 lacks the J domain responsible for stimulating DnaK
ATPase activity. Therefore, CYO1 may function without Hsp70s.
DnaJ-like proteins play an important role in plastid biogenesis.
The cauliflower (Brassica oleracea) OR gene encodes a DnaJ
Cys-rich domain–containing protein that participates in the differentiation of chromoplastids for carotenoid accumulation (Lu
et al., 2006); however, the function of OR has yet to be elucidated. CYO1 and OR are present only in higher plants, not in
moss and algae. CYO1 (At3g19220) shares only 13% identity
with the Arabidopsis OR homolog At5g61670. Maize BSD2 also
has a Cys-rich zinc binding domain similar to that in DnaJ and is
targeted to chloroplasts (Brutnell et al., 1999). BSD2 is not involved in general photosynthetic complex assembly or protein import but is required for the posttranslational regulation of RBC-L.
CDJ2, a chloroplast DnaJ homolog, may function during the biogenesis and/or maintenance of thylakoid membranes (Liu et al.,
2005). This protein has the J domain that interacts with Hsp70
but lacks a Cys-rich domain. RB60 is an atypical PDI that functions as a member of a redox regulatory protein complex controlling translation in Chlamydomonas chloroplasts (Kim and
Mayfield, 1997). RB47, a member of the eukaryotic poly(A)
binding protein family, binds directly to the 59 untranslated region
of psbA mRNA, which encodes the photosynthetic reaction
center protein D1 in chloroplasts. RB60 binds to RB47 and
modulates its activity via redox and phosphorylation. Unlike
CYO1, these proteins are soluble (Yohn et al., 1996), suggesting
that CYO1 is a novel PDI in the chloroplast thylakoid membrane
that acts as a chaperone-like factor for thylakoid membrane
proteins.
Many thylakoid membrane proteins contain Cys residues.
Recently, Motohashi and Hisabori (2006) reported that several
thylakoid membrane proteins interact with HCF167, a thioredoxinlike protein in thylakoids. All of the proteins they identified, including cytochrome f, Rieske FeS protein, PSI-N, LHCB5, FTSH2,
FTSH8, and three CF1 subunits of ATPase, have multiple Cys
residues. CYO1 behaves as an intrinsic thylakoid membrane
protein and may interact with the PSI/LHCI and PSII/LHCII
complexes (Figure 6E). Many hydrophobic subunits of PSI and
PSII have Cys residues (Table 1). Because the initial phase of
chloroplast biogenesis in the cotyledon is characterized by the
rapid synthesis and assembly of the photosynthetic thylakoid
membrane system, the PDI activity of CYO1 may be involved
directly in disulfide bond formation and/or accelerating the
folding of Cys-rich proteins (e.g., hydrophobic subunits of PSI
and PSII). This hypothesis requires further investigation.
Cotyledons switch from sink to source status during development in order to support chloroplast biogenesis in the leaf, which
is essential for survival in the field. Consequently, chloroplast
biogenesis in cotyledons occurs when energy is limited. Therefore, rapid synthesis and assembly of the photosynthetic thylakoid membrane system is critical. CYO1 may be involved directly
in this process. In cyo1 mutants, the period between germination
and development of the first leaf is longer and the growth rate is
slower than in wild-type plants. Consequently, plants that harbor
CYO1 may have a selective advantage compared with those that
lack this gene.
METHODS
Plant Growth and Isolation of the Mutant
Arabidopsis thaliana (ecotype Wassilewskija) was grown at 238C under
continuous light on 1/2 MS plates or on soil. Transgenic plants were
produced on a large scale by vacuum infiltration (Bechtold and Pelletier,
1998), with Agrobacterium tumefaciens strain GV310 (pmp90rk) harboring the activation-tagging vector pPCVICEn4HPT (Hayashi et al., 1992;
Shirano et al., 2000). We isolated several mutants with white cotyledons
from among ;3500 transgenic lines. The mutant line designated cyo1
was chosen for further study.
Microscopy
Plant tissues were fixed with glutaraldehyde and paraformaldehyde and
embedded in Quetol 812 (Nisshin EM) according to standard procedures.
Ultrathin sections were stained with uranyl acetate, then with lead citrate,
and observed with a JEM 1200EX transmission electron microscope.
Isolation of the T-DNA–Flanking Sequence
The T-DNA fragment used for Arabidopsis transformation contained the
Col E1 origin and the ampicillin resistance gene (Hayashi et al., 1992) and
was isolated by plasmid rescue in Escherichia coli. Genomic DNA was
extracted from cyo1 mutant plants as described by Saghai-Maroof et al.
(1984), digested with ClaI, and then ligated and transformed into E. coli
XL2-Blue. The rescued plasmid containing the T-DNA–flanking fragment
was sequenced.
RNA Expression Analyzed by RT-PCR
CYO1 gene expression levels were assessed by a kinetic RT-PCR approach. 59-ATGTTCCGATTATACCCTAATTGCTCTCTG-39 and 59-TATCCAAATCTTCCATCACTTCAATGTCCG-39 were used as CYO1-specific
primers, and 59-CTTCGTCAAGACTTTGACCG-39 and 59-GCCCCAAAACACAAACCACC-39 were used as a control for constitutive expression
(UBQ10) (Awai et al., 2001). All genes tested are unique in the Arabidopsis
genome and show the same linear amplification rate from 13 to 21 cycles
(data not shown). Single-stranded DNA was synthesized from total RNA
Arabidopsis Protein Disulfide Isomerase CYO1
(0.25 mg) using avian myeloblastosis virus RT (TaKaRa) in the presence of
oligo(dT) primer at 558C for 30 min. PCR aliquots (19th cycle) were analyzed on agarose gels, blotted, and radiolabeled with specific probes
corresponding to the amplified fragments.
3167
(Bruce et al., 1994). Intact pea chloroplasts were resuspended in the
import buffer (330 mM sorbitol and 50 mM HEPES/KOH, pH 8.0) at a
concentration of 1 mg chlorophyll/mL. Import reactions were performed
as described by Bruce et al. (1994). Import and extraction experiments
were performed as described previously by Tranel et al. (1995).
Immunoblot Analysis
Rabbit antiserum to recombinant full-length CYO1 protein was prepared
by a conventional method (Suzuki et al., 2002). Intact chloroplasts of 7-dold Arabidopsis grown under continuous light were isolated according to
the method of Aronsson and Jarvis (2002). Briefly, plants were homogenized and the homogenate was filtered through a double layer of
Miracloth (Calbiochem). The filtrate was used as total protein (Figure
6B). The filtrate was centrifuged at 1000g for 5 min, and the pellet was
resuspended. The resuspended chloroplasts were loaded onto a twostep Percoll gradient (40 and 85% Percoll) and centrifuged at 2500g for
10 min. The intact chloroplasts that appeared between the phases were
washed and used as intact chloroplasts (Figure 6B). The intact chloroplasts were lysed osmotically by suspending them with 50 mM Tris-HCl,
pH 7.5, vortexed vigorously, and then centrifuged at 3000g for 5 min.
The pellet was washed twice and used as the pellet sample (Figure 6B).
The supernatant was centrifuged at 10,000g for 30 min and used as the
supernatant sample (Figure 6B). The proteins were electrophoresed on a
12.5% SDS-PAGE gel and electroblotted onto nitrocellulose. The membrane was immunoreacted with each antibody. Immunoreactive proteins
were detected using the ECL Advance Western Blotting Detection Kit (GE
Healthcare) and Kodak Image Station 2000R for CYO1 or the Alkaline
Phosphatase Substrate Kit II (Vector Laboratories) for other proteins.
Intact thylakoids from Arabidopsis were prepared as described
(Casazza et al., 2001). Sonicated thylakoids were prepared by sonicating
thylakoid membranes for 3 min at 08C using an ultrasonic disruptor
(TOMY, UD-201, output 2, duty cycle 30). For alkaline, detergent, and
high sodium chloride treatments, the sonicated thylakoids were resuspended with 100 mM sodium carbonate, pH 11.5, 1.0% Nonidet P-40, or
2.0 M sodium chloride in the extraction buffer. After incubation for 1 h on
ice, samples were centrifuged at 10,000g for 10 min at 08C to separate
soluble and insoluble fractions. A protease protection assay was performed as described (Motohashi and Hisabori, 2006).
Blue native gel electrophoresis was performed as described (Asakura
et al., 2004) (see also http://www.hos.ufl.edu/clineweb/BNgel.htm) with
the following modifications. Intact thylakoids (0.8 mg chlorophyll/mL)
were solubilized with 12.5 mM BisTris-HCl, pH 7.0, 10% (w/v) glycerol,
and 1% (w/v) n-dodecyl-b-D-maltoside (Dojindo) and incubated for
30 min at 48C. Samples were centrifuged at 15,000g for 30 min at 48C.
One hundred microliters of the supernatant was combined with 10 mL of
blue native sample buffer (5% Coomassie Brilliant Blue G 250 [CBB G
250], 100 mM BisTris-HCl, and 0.5 M 6-amino-capronic acid, pH 7.0).
Fifteen microliters of sample (corresponding to 5.5 mg of chlorophyll) was
loaded onto a blue native 5 to 14% polyacrylamide gradient gel. Electrophoresis was performed at 100 V at 48C. The cathode buffer initially
contained 0.01% CBB G 250 and was replaced by buffer lacking CBB G
250 after approximately half of the electrophoresis run. After electrophoresis, the gel was incubated with blotting buffer (100 mM Tris, 192 mM
Gly, and 5% methanol) containing 0.1% SDS for 10 min at room
temperature. The proteins in the gel were electroblotted onto a nitrocellulose membrane filter using blotting buffer.
In Vitro Chloroplast Import Assay
CYO1 protein and RBC-S (Olsen and Keegstra, 1992) were translated and
radiolabeled using [35S]Met and the TNT coupled wheat germ extraction
system (Promega) according to the manufacturer’s protocol. Intact
chloroplasts were isolated from 8- to 12-d-old pea (Pisum sativum)
seedlings and purified over a Percoll gradient as described previously
RNA Gel Blot Analysis
Total RNA was prepared using the RNeasy mini kit (Qiagen) according to
the directions supplied. Total RNA (1.0 mg) was electrophoresed on a
1.2% agarose/formaldehyde gel and blotted onto nylon membranes. The
membranes were hybridized to random-primed [32P]cDNA probes. After
hybridization, the membranes were washed with 0.13 SSC (13 SSC is
0.15 M NaCl and 0.015 M sodium citrate)/0.1% SDS at 658C for 60 min
and autoradiographed.
Expression and Purification of CYO1 and DnaJ Proteins
The full-length CYO1 cDNA was amplified by RT-PCR using primers that
contained sites for NdeI (CYO1-5-Nde, 59-CCCCATATGTTCCGATTATACCCTAATTGCTCTCTG-39) and HindIII (CYO1-3-Hin, 59-GGGAAGCTTAGATGGTTCATTATCCAAATCTTCCATCAC-39). Similarly, a primer
(CYO1-31aa, 59-CCCCATATGGCCGCCGCTGATATTCCCCTTGGTGACGGA-39) was constructed, and the primer set CYO1-31aa and
CYO1-3-Hin was used for PCR. The amplified DNA fragments encoded
the D30CYO1 protein containing amino acids 31 to 187. The DnaJ gene in
E. coli was amplified by PCR using primers containing sites for NdeI
(EdnaJ-5Nde, 59-GGGCATATGGCTAAGCAAGATTATTACGAGATTTTA-39)
and XhoI (EdnaJ-3-Xho, 59-GGGCTCGAGGCGGGTCAGGTCGTCAAAAAACTTCTTCAC-39). The amplified DNA fragments were digested
with NdeI and HindIII or XhoI and ligated into the expression vector
pET-24a(þ) (Novagen). The sequences of the amplified fragments were
confirmed. Expression and purification were performed as described
previously (Shimada et al., 2004). E. coli strain BL21(DE3) was used for the
expression of recombinant fusion proteins bearing a 6xHis tag. Overnight
cultured E. coli BL21(DE3) harboring the full-length cDNA of the CYO1
gene was diluted 1:10 with fresh culture medium and further grown at
378C for 1 h. Isopropyl b-D-thiogalactoside was then added to a final
concentration of 1.0 mM, and the cells were further cultured at 378C
overnight. The cells were centrifuged for 10 min at 5000g and 48C. The cell
pellets were suspended in suspension buffer (15.0 mM Tris-HCl, pH 7.0,
50.0 mM NaCl, 0.1 mM EDTA, 0.1 mM DTT, and 1.0 mM phenylmethylsulfonyl fluoride). The crude extracts were sonicated and centrifuged for
10 min at 12,000g and 48C. The pellets were suspended in suspension
buffer supplemented with 6.0 M guanidine hydrochloride and sonicated.
The samples were incubated overnight with nickel-nitrilotriacetic acid
agarose (Qiagen) at 48C. The agarose was washed with suspension buffer
supplemented with 6.0 M guanidine hydrochloride and then washed with
suspension buffer supplemented with 8.0 M urea. Proteins bound to the
agarose were eluted with suspension buffer containing 8.0 M urea and
50.0 mM imidazole. The eluted proteins were dialyzed with renaturation
buffer (15.0 mM Tris-HCl, pH 6.3, and 50.0 mM NaCl). The dialyzed
samples were centrifuged for 10 min at 12,000g and 48C. The pellet was
suspended with renaturation buffer, and the proteins were injected into
rabbits to stimulate antibody production.
E. coli strain BL21(DE3) was used for the expression of recombinant
D30CYO1 and DnaJ fusion proteins with 6xHis tags. Overnight cultured
BL21(DE3) harboring the D30CYO1 or DnaJ gene was diluted 1:20 with
fresh culture medium and further grown at 378C for 1 h. Isopropyl b-Dthiogalactoside was then added to a final concentration of 1.0 mM,
and the cells were further cultured at 308C (D30CYO1) or 208C (DnaJ)
overnight. The cells were centrifuged for 10 min at 5000g and 48C. The cell
pellets were suspended in suspension buffer. The crude extracts were
sonicated and centrifuged for 10 min at 12,000g and 48C. The pellets were
3168
The Plant Cell
suspended in suspension buffer supplemented with 6.0 M guanidine
hydrochloride and sonicated. Then, the samples were dialyzed with
dialyzing buffer (15 mM Tris-HCl, pH 7.0, and 50 mM NaCl). The samples
were centrifuged for 10 min at 12,000g and 48C, and the supernatant was
incubated for 3 h with nickel-nitrilotriacetic acid agarose (Qiagen) at 48C.
The agarose was washed with dialyzing buffer containing 50 mM imidazole. Proteins bound to the agarose were eluted with dialyzing buffer
containing 150 mM imidazole. The eluted proteins were denatured with
6.0 M guanidine hydrochloride, and then the proteins were renatured with
dialyzing buffer containing 5.0 mM ZnCl2. To analyze the requirement of
Zn for the catalytic properties of CYO1, the proteins were renatured with
dialyzing buffer containing 5.0 mM ZnCl2, CuCl2, CaCl2, MgCl2, or no
divalent metal ion. The samples were centrifuged for 10 min at 12,000g
and 48C. The supernatants were used for enzymatic assays.
Assay of Insulin Disulfide Reduction
The reduction of insulin (Sigma-Aldrich) was assayed by measuring the
increase in absorbance at 650 nm (Holmgren, 1979).
f; Maryse A. Block (Centre National de la Recherche Scientifique/Institut
Recherche et Developpement/Universite de Perpignan, France) for the
gift of antibodies against E37; Yukiko Sasaki and Kan Tanaka (University
of Tokyo) for the gift of antibodies against ACCD; Masayoshi Maeshima
(Nagoya University) for the gift of antibodies against PAQ; and Ikuko
Hara-Nishimura (Kyoto University) for the gift of antibodies against BiP.
We are also grateful to Yuji Saito (Tokyo Institute of Technology) for
technical advice on the assay for PDI activity and to Toru Hisabori,
Ken Motohashi, Hiroyuki Ohta (Tokyo Institute of Technology), Tatsuru
Masuda (University of Tokyo), and Hirofumi Kuroda (RIKEN Plant Science
Center) for valuable discussions. This study was partially supported by
the Ministry of Education, Science, Sports, and Culture (Grant-in-Aid for
Scientific Research C, 16770030, to H.S.) and by a grant from the U.S.
National Science Foundation (to K.W.O).
Received March 19, 2007; revised September 13, 2007; accepted September 19, 2007; published October 5, 2007.
Assay of PDI Activity
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Accession Number
Sequence data from this article can be found in the Arabidopsis Genome
Initiative database under accession number At3g19220 (CYO1).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Levels of Chlorophylls a and b.
Supplemental Figure 2. Ultrastracture of Chloroplasts of cyo1 in
Rosette Leaves.
Supplemental Figure 3. Genomic DNA Gel Blot Analysis of T-DNA in
cyo1.
Supplemental Figure 4. A Ten-Day-Old cyo1 Mutant Plant Harboring
a Vector Containing the cDNA between 5 and 606 bp with the ATG at
32 bp Mutated to ATT.
Supplemental Figure 5. Comparison of mRNA Levels for 2880 ESTs
in CYO1 Mutant and Wild-Type Plants.
ACKNOWLEDGMENTS
We dedicate this article to Ken-ichiro Takamiya, who passed away in an
unfortunate traffic accident while this study was being conducted. We
thank Toru Hisabori (Tokyo Institute of Technology) for the gift of
antibodies against PSI-N, FNR, TRX-M, CF1-b, and CF1-g; Amane
Makino (Tohoku University) for the gift of antibodies against cytochrome
Arabidopsis Protein Disulfide Isomerase CYO1
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modification of nascent secretory proteins. Cell 57: 1069–1072.
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Arabidopsis Cotyledon-Specific Chloroplast Biogenesis Factor CYO1 Is a Protein Disulfide
Isomerase
Hiroshi Shimada, Mariko Mochizuki, Kan Ogura, John E. Froehlich, Katherine W. Osteryoung, Yumiko
Shirano, Daisuke Shibata, Shinji Masuda, Kazuki Mori and Ken-ichiro Takamiya
Plant Cell 2007;19;3157-3169; originally published online October 5, 2007;
DOI 10.1105/tpc.107.051714
This information is current as of June 16, 2017
Supplemental Data
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References
This article cites 47 articles, 23 of which can be accessed free at:
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