Shinji Kawasaki*, Keisuke Mizuguchi, Masaru Sato, Tetsuya Kono and Hirofumi Shimizu
Department of Biosciences, Tokyo University of Agriculture, Setagaya-ku, Tokyo, 156-8502 Japan
*Corresponding author: E-mail, [email protected]; Fax, +81-3-5477-2764.
(Received April 16, 2013; Accepted May 24, 2013)
Water-soluble orange carotenoid proteins (OCPs) that
bind 30 -hydroxyechinenone are found in cyanobacteria,
and are thought to play a key role in photoprotection. The
distribution of OCPs in eukaryotes remains largely unknown.
In this study, we identified a novel OCP that predominantly
binds astaxanthin from a eukaryotic microalga, strain Ki-4,
isolated from a dry surface of heated asphalt in midsummer.
A purified astaxanthin-binding OCP, named AstaP, shows
high solubility in water with an absorption peak at
484 nm, and possesses a heat-stable activity that quenches
singlet oxygen. The deduced amino acid sequence of AstaP
comprises an N-terminal hydrophobic signal peptide, fasciclin domains found in secreted and cell surface proteins, and
N-linked glycosylation sites, the first example of a carotenoprotein among fasciclin family proteins. AstaP homologs of
unknown function are distributed mainly in organisms from
the hydrosphere, such as marine bacteria, cyanobacteria, sea
anemone and eukaryotic microalgae; however, AstaP exhibits a unique extraordinarily high isoelectric point (pI)
value among homologs. The gene encoding AstaP, as well
as the AstaP peptide, is expressed abundantly under conditions of dehydration and salt stress in conjunction with high
light exposure. As a unique aqueous carotenoprotein, AstaP
will provide a novel function of OCPs in protection against
extreme photooxidative stresses.
Keywords: Astaxanthin Carotenoid Fasciclin Microalgae
Photooxidative stress Singlet oxygen.
Strain Ki-4 has been deposited in the National Biological
Resource Center (NBRC) at the National Institute of
Technology and Evaluation (NITE) as strain number
NBRC108794. The 18S rRNA gene sequence and the ITS gene
Introduction
Under extreme environments such as desiccation and high salinity combined with high light irradiation, it is difficult for
higher plants to survive; however, microalgae are found to
thrive under such conditions in which some unknown mechanisms to protect against photooxidative stress must be functioning. Light energy, in combination with oxygen, leads to the
generation of reactive oxygen species under water stress conditions (Mehler 1951, Ananyev et al. 1994). Plants are known to
possess antioxidant enzymes such as superoxide dismutase and
ascorbate peroxidase that scavenge superoxide anions and
hydrogen peroxide in chloroplasts (Asada 1999, Cheeseman
2007, Foyer and Shigeoka 2011). Plants also use carotenoids,
including those of the xanthophyll cycle, which is localized in
the light-harvesting antenna complexes in the thylakoid membrane, to dissipate excess light energy by quenching Chl triplet
states, thus preventing the formation of singlet oxygen (1O2)
(Telfer et al. 2008, Jahns and Holzwarth 2012). 1O2 is also produced by photosensitization of biomolecules such as porphyrins and flavins, and prefers to oxidize unsaturated lipids in the
cell membrane, causing, first, lipid peroxidation and, ultimately,
functionally significant damage to membranes, proteins and
nucleic acids through a radical chain reaction (Halliwell and
Chirico 1993, Blokhina et al. 2003).
Although >700 fully characterized, naturally occurring carotenoids are hydrophobic, a few are known to be naturally water
dispersible due to the presence of binding sugars that give a typical surfactant structure (Sliwka et al. 2010), or to dissolve in
aqueous solution through binding proteins (Holt and
Krogmann 1981, Kerfeld et al. 2010). The aqueous carotenoproteins, known as orange carotenoid binding proteins (OCPs), that
bind 30 -hydroxyechinenon and are widely distributed in cyanobacteria (Holt and Krogmann 1981), have been well characterized
Plant Cell Physiol. 54(7): 1027–1040 (2013) doi:10.1093/pcp/pct080, available FREE online at www.pcp.oxfordjournals.org
! The Author 2013. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
Plant Cell Physiol. 54(7): 1027–1040 (2013) doi:10.1093/pcp/pct080 ! The Author 2013.
Editor-in-Chief’s choice
Abbreviations: BSA, bovine serum albumin; CFE, cell-free extract; 2-D, two-dimensional; ITS, internal transcribed spacer; LC,
liquid chromatography; MS, mass spectrometry; NMR, nuclear
magnetic resonance; OCP, orange carotenoid protein; PAS,
Periodic acid–Schiff; pI, isoelectric point; RACE, rapid amplification of cDNA ends; SOSG, Singlet Oxygen Sensor Green
sequence of strain Ki-4, and cDNA and genome sequences
encoding AstaP have been deposited in the DDBJ/EMBL/
Genbank database under the accession numbers AB734096,
AB762691, AB731756 and AB731757, respectively.
Rapid Paper
A Novel Astaxanthin-Binding Photooxidative Stress-Inducible
Aqueous Carotenoprotein from a Eukaryotic Microalga
Isolated from Asphalt in Midsummer
1027
S. Kawasaki et al.
in terms of function and structure (Kerfeld et al. 2003, Kerfeld
et al. 2010). Based on their high light-inducible expression profiles
(Hihara et al. 2001) and their role in light energy dissipation, the
functions of OCPs are proposed to involve photoprotection by
interacting with phycobilisome (Wilson et al. 2006, Kerfeld et al.
2010, Gwizdala et al. 2011, Berera et al. 2012, Wilson et al. 2012,
Kirilovsky and Kerferd 2013). In eukaryotes, no homologs of
cyanobacterial OCPs have been found in the genome of
Chlamydomonas and higher plants (Kerfeld et al. 2003). An exception, to our knowledge, is a yellow water-soluble carotenoprotein purified from Scenedesmus obliquus D3 that binds
predominantly to violaxanthin and, to a lesser extent, to neoxanthin (Powls and Britton 1976).
In this study, we report the discovery from a eukaryotic
microalga of a soluble OCP that predominantly binds astaxanthin. Astaxanthin is a lipophilic carotenoid that imparts an
attractive red-orange color to the muscle of salmon, the feathers of birds such as flamingoes (Fox 1955), the petals of the
adonis flowers (Cunningham and Gantt 2011) as well as the
blue color to the carapace of lobsters and other crustaceans
(Britton and Helliwell 2008). Astaxanthin has attracted attention in the medical, cosmetic and healthcare industries due to
its powerful antioxidative properties. More recently, astaxanthin has been recognized as a valuable carotenoid in
humans based on its potential roles in the prevention of eye,
skin, inflammatory, neural and blood diseases, and cancer
(Guerin et al. 2003, Hussein et al. 2006, Higuera-ciapara et al.
2006, Kidd 2011, Fassett and Coombes 2012). Astaxanthin is
distributed in these organisms by binding to proteins such as
a- and b-crustacyanin in shells, the actomyosin complex of
muscle or keratin in feathers, and localizes mainly at the external surface of the organism (Britton and Helliwell 2008).
Because animals do not synthesize astaxanthin de novo,
microalgae serve as their natural dietary source. The chlorophyte microalga Haematococcus pluvialis is thought to
accumulate the highest levels of astaxanthin in nature
(Boussiba 2000), and is cultivated as a natural source of astaxanthin for industry. Haematococcus astaxanthin is readily esterified and extracted as a lipophilic mono- or di- esterified form;
it is synthesized as a secondary carotenoid and accumulates
in extraplastidic lipid vesicles (Boussiba 2000, Collins et al.
2011, Wayama et al. 2013). The photoprotective role of Haematococcus astaxanthin is proposed to be based on its sunshade effect (Yong and Lee 1991, Hagen et al. 1994) as well as
on its efficient ability to quench 1O2 (Di Massio et al. 1990,
Kobayashi and Sakamoto 1999).
We also report the identification of novel functions including
carotenoid binding and antioxidative activity among fasciclin
family proteins. Fasciclin family proteins possess fasciclin domains that are conserved in secretory and membrane-anchored
proteins, and are reported to localize at the cell surface and to be
involved potentially in biological functions such as cell adhesion,
cell proliferation, cell expansion, tumor development and plant
reproduction (Zinn et al. 1988, Skonier et al. 1992, Takeshita et al.
1993, Huber et al. 1994, Kim et al. 2002, Clout et al. 2003, Shi et al.
1028
2003, Johnson et al. 2003, Kii and Kudo 2006, Ruan et al. 2009, Li
et al. 2010, Kudo 2011, Tan et al. 2012).
Here we report the purification and characterization of
a novel astaxanthin-binding protein from a eukaryotic microalga isolated from an extreme environment. This unique aqueous carotenoprotein appears to provide a novel way of
protecting unicellular organisms from extreme photooxidative
stresses.
Results and Discussion
Characterization of strain Ki-4 isolated from an
extreme environment
Strain Ki-4 was isolated from a dried surface of heated asphalt in
front of Tokyo University of Agriculture in midsummer, 2003. A
single colony developed under sunlight was chosen and cultivated. Strain Ki-4 was selected from among 40 isolates based on
its ability to tolerate dehydration stress under high light exposure conditions.
Strain Ki-4, a unicellular eukaryotic microalga, has cells that
are oval in shape with polar thickening, ornamented with meridional ribs, 5–10 mm in length and 3–8 mm in width, and are
non-flagellar (Fig. 1A). During cultivation in both liquid and
solid media, the strain is always observed as unicellular bodies
without the formation of coenobia. The 18S rRNA gene and
internal transcribed spacer (ITS) gene sequences revealed that
the strain is classified in the family Scenedesmaceae of the order
Chlorococcales, and the phenotypic and phylogenetic characteristics show a close relationship to Scenedesmus strains isolated from desert environments (Lewis and Flechtner 2004)
(Supplementary Fig. S1). According to the literature that includes the recent classification of Scenedesmaceae (Krienitz and
Bock 2012), strain Ki-4 shows similar morphological characteristics to the genus Coelastrella, but strain Ki-4 does not form
2- or 4-celled groups.
Strain Ki-4 survives under desiccated and high light
conditions for >6 months (Fig. 1B). As the water content
of the agar plate is decreased to <1% over 8 d, the cells
gradually change in color from green to red-orange (Fig. 1B).
The strain also grows well in liquid medium. Although the
strain prefers non-salty medium, it shows salt tolerance
(up to about 1 M NaCl) under high light conditions, with
a change in color from green to orange (Fig. 1B). A Chlamydomonas reinhardtii strain changes in cell color progressively
from green to white under these stress conditions (Fig. 1C).
Based on these ecological and physiological properties, we
anticipated that the strain Ki-4 would possess outstanding abilities to protect a single cell under extreme photooxidative stress
conditions.
Purification of an aqueous orange carotenoid
protein
The expression of an aqueous OCP was first found in cell
extracts of salt-stressed cells under high light conditions
Plant Cell Physiol. 54(7): 1027–1040 (2013) doi:10.1093/pcp/pct080 ! The Author 2013.
Astaxanthin binding orange carotenoid protein
A
1
2
3
A
2.0
1.5
5 µm
5 µm
Abs
484
10 µm
1.0
0.5
B
0
250
Dehydration
0 day w/HL
Dehydration
8 days w/HL
B
400
nm
600
800
600
800
1.5
484
Abs
1.0
non stress
0 day w/HL
dehydration w/HL
1 month
6 months
283
0.5
rehydration
3 days
0
250
400
nm
non stress
0 day w/HL
0.9 M NaCl
6 days w/HL
C
C
(kDa)
250
150
100
75
1
2
50
37
Dehydration
0 day w/HL
25
20
Dehydration
6 days w/HL
15
10
0 M NaCl
24 hours w/HL
0.5 M NaCl
24 hours w/HL
Fig. 1 Morphology and stress tolerance of strain Ki-4. (A) 1, Optical
(phase contrast); and 2 and 3, scanning electron micrographs of strain
Ki-4. Scale bars are as indicated in the figure. (B) Survival of strain Ki-4
under dehydration and salt stress conditions with high light (w/HL).
When fresh medium is soaked onto the plate following 6 months of
dehydration, the red-orange cells change to green (middle panel). (C)
Clamydomonas reinhardtii NIES2238 changed in cell color from green
to white under dehydration (upper panel) and salt stress (lower panel)
conditions with high light.
for 6 d (Fig. 2A). An orange supernatant obtained after
ultracentrifugation of cell extracts at 100,000g was further
purified by passage through CM-Sepharose and gel filtration columns. Elution yielded a single orange peak that
appeared as a single band with an apparent molecular mass
of 33 kDa on SDS–PAGE (Fig. 2C); the apparent molecular
mass of the native protein was estimated to be approximately
42–43 kDa by gel filtration chromatography (Supplementary
Fig. S2). These data demonstrate that the protein is a
monomer. The purified orange protein shows absorption
maxima at 283 and 484 nm (A484/A283 = 1.8) in ultra-pure
water (Fig. 2B).
Fig. 2 Purification of an aqueous orange carotenoid protein. (A)
Spectrum of the CFE of stressed Ki-4 cells after ultracentrifugation.
A 1.0 g aliquot of wet cells stressed by 0.7 M salt under high light
conditions for 6 d was dissolved in 9.0 ml of 50 mM Tris–HCl buffer,
pH 7.5, disrupted in a French press and then ultracentrifuged. The
inset shows the aqueous supernatant after ultracentrifugation of nonstressed (left tube) and salt-stressed (right tube) CFEs. (B) Spectrum of
the purified AstaP after passage through a desalting column PD-10
eluted with MilliQ ultra-pure water. The inset shows the purified
AstaP. (C) SDS–PAGE of the purified AstaP stained with Coomassie
Brilliant Blue. The protein standards (lane 1) and the purified protein
(lane 2) are indicated along with their corresponding molecular
masses (kDa).
Determination of the binding pigment of AstaP
The binding pigment of AstaP was determined based on the
absorption spectra obtained using an HPLC photodiode array
detector, HPLC retention times and molecular masses in highresolution liquid chromatography/mass spectrometry (LC/MS)
analysis in comparison with standard compounds (Fig. 3;
Supplementary Fig. S3). The major binding pigment P1 was
determined to be astaxanthin, which was confirmed by 1H-nuclear magnetic resonance (NMR) (Supplementary Fig. S4). The
minor peak P2 shows the same mass spectrum as 4-ketozeaxanthin (adonixanthin), which is a known astaxanthin precursor,
Plant Cell Physiol. 54(7): 1027–1040 (2013) doi:10.1093/pcp/pct080 ! The Author 2013.
1029
S. Kawasaki et al.
AstaP
480 nm
P1
mAU
A
P2
P3
P4
Retention time (min)
Standards
mAU
B
Astaxanthin
Adonixanthin
Lutein Canthaxanthin
480 nm
Retention time (min)
478
464
445
473
476
mAU
C
P1: RT = 14.8 min
P3: RT = 19.8 min
468
445
473
mAU
478
P2: RT = 17.5 min
Astaxanthin: RT = 14.8 min Adonixanthin: RT = 17.4 min
Lutein: RT = 19.8 min
P4: RT = 23.2 min
nm
476
nm
Canthaxanthin: RT = 23.2 min
Fig. 3 Determination of carotenoids bound to AstaP using an HPLC photodiode array detector. (A) Elution profiles of the binding pigments of
AstaP. (B) Elution profiles of authentic standards. (C) Spectrum of each elution peak in A and authentic standards in B. HPLC retention time (RT)
is shown below the spectrum. High-resolution LC/MS data are shown in Supplementary Fig. S3.
but the HPLC retention time and the UV/Vis spectrum differ
slightly from those of synthetic standard adonixanthin (tentatively named carotenoid-x). The minor peaks P3 and P4 were
determined to be lutein and canthaxanthin. The binding ratios
of astaxanthin (P1), carotenoid-x (P2), lutein (P3) and canthaxanthin (P4) were estimated to be 80, 14, 3 and 3%, respectively.
AstaP is a fasciclin-like glycosylated protein
The N-terminal amino acid sequence of the purified AstaP was
determined to be ATPKAXATTAKPASTTSTPVYATLSNAVTA.
An internal peptide sequence (ALKDXATVATALKDASVTV)
was obtained from a peptide fragment by trypsin digestion.
The residues represented by X in both fragments showed no
signal on peptide sequencing (Supplementary Fig. S5), suggesting the existence of some chemical modification at these
sites. Degenerate PCR using primers containing a mixed base
derived from amino acid sequences yielded a cDNA fragment
clone; the full-length cDNA encoding AstaP (astaP) was
1030
obtained by 50 -rapid amplification of cDNA ends (RACE)
PCR. The deduced amino acid sequence from astaP yielded
an N-terminal hydrophobic signal sequence, two fasciclin-like
H1 and H2 domains (Kawamoto et al. 1998) and five putative
N-glycosylation Asn-x-Thr sites (Fig. 4A).
A homology search using the BLAST and FASTA programs
found proteins of unknown function (Fig. 5), a large majority of
which are from organisms living in or isolated from the hydrosphere, such as marine bacteria, cyanobacteria, a sea anemone
Nematostella vectensis and the eukaryotic microalgae Senedesmus acutus, Chlorella variabilis and C. reinhardtii; no homologs
from higher plants were found. The top hit in both search
results was a protein named ‘fasciclin domain-containing protein’ from the marine bacterium Algoriphagus sp. PR1 reported
as a carotenoid-producing pink-colored bacterium (Alegado
et al. 2011). This homologous protein shows 43% identity in
overlapping regions (accession No. ZP_07718665), contains
an N-terminal signal sequence and lacks N-glycosylation sites
Plant Cell Physiol. 54(7): 1027–1040 (2013) doi:10.1093/pcp/pct080 ! The Author 2013.
Astaxanthin binding orange carotenoid protein
A
MRRNSIVLLVLFCLVALATAATPKANATTAKPASTTSTPVYATLSNAVTAGAAAPQL
TTLFAAVRAANVTGALTANTTWTILAPTNDAFAKRLAKLNLTADAVLKNKDLLVKIL
SYHVIPSGAVYSKALKDNATVATALKDASVTVRLYQGKVMFKGPVNKAQVTVADIKA
GGSVIHVINDVLLPPGVVSDAVAKQWKAEWEAMKAEKKVAPKATTGRRFLLF
CBB stained
C
B
1
ATG
TGA
500 bp
2
3
PAS stained
4
1
(kDa)
(kDa)
250
150
100
75
250
150
100
75
50
37
50
37
25
20
25
20
15
15
10
10
2
3
4
Fig. 4 AstaP belongs to the fasciclin-like glycosylated protein family. (A) The deduced amino acid sequence of AstaP. The arrow indicates the
cleavage site for the N-terminal signal peptide (underlined with dots). H1 and H2 motifs are shown in bold italics. Potential sites for N-linked
glycosylation are boxed, and an experimentally predicted glycosylated asparagine is in bold in the boxes. YH and DI motifs, which are shown in
bold, are highly conserved in fasciclin family proteins, and reported to interact with integrins (Kim et al. 2002). (B) Structure of the astaP gene.
The start and stop codons are indicated. Filled boxes represent exons, and the lines between filled boxes represent introns. (C) PAS staining of the
purified AstaP. Lane 1, the pre-stained protein standards with molecular masses (kDa); lane 2, purified AstaP; lane 3, BSA (Sigma); lane 4,
glycosylated bovine fetuin (Sigma, the molecular mass of non-glycosylated fetuin is 48.4 kDa).
(Fig. 5A). The gene sll1483 in Synechocystis sp. PCC 6803 encodes a homolog (38% identity in overlapping regions) that is
annotated as ‘transforming growth factor-induced protein’, and
contains an N-terminal signal sequence and fasciclin domains
(Fig. 5). It was identified by microarray analysis as one of the upregulated genes within 15 min after the shift of light intensity
from low light (20 mmol photons m2 s1) to high light
(300 mmol photons m2 s1) (Hihara et al. 2001). Functional
properties of bacterial homologs are now being investigated
in our laboratory. A Scenedesmus homolog (accession No.
ACB06751) of unknown function, which shows 40% identity
in overlapping regions, possesses fasciclin domains and does
not contain N-glycosylation sites, and is reportedly induced
by Cr stress; a cell-surface localization has been suggested (Torelli et al. 2008) (Fig. 5). Most of these homologs exhibit an acidic
isoelectric point (pI.4–6), with the exception of those from
Meiothermus ruber (pI = 9.4) and Isosphaera pallida (pI = 9.0),
which are known as carotenoid-producing bacteria (Giovannoni et al. 1987, Burgess et al. 1999). AstaP is unique in
having an extraordinarily high pI of 10.5 (Fig. 7B). The H1
and H2 domains found in these fasciclin family proteins are
conserved in secretory and membrane-anchored proteins
such as fasciclin I from Drosophila (Zinn et al. 1988), big-h3
(Skonier et al. 1992) and periostin (Takeshita et al. 1993)
from human, arabinogalactan proteins from plants (Schultz
et al. 2000, Shi et al. 2003), algal cell adhesion molecules
(CAMs) from Volvox (Huber and Sumper 1994) and several
fungal, yeast and bacterial proteins of unknown function
(Figs. 5, 6). To our knowledge, none of these proteins is reported to bind carotenoids or to possess an antioxidative function. N-terminal hydrophobic signal sequences are highly
conserved in these proteins (Figs. 5A, 6). An N-terminal hydrophobic peptide was also detected in AstaP, and the cleavage site
was determined to lie between Ala20 and Ala21 based on the
N-terminal amino acid sequence of the purified protein
(Fig. 4A), which coincides with the predictions made by the
PSORT, SignalP and TargetP programs. The localization of AstaP
is suggested to lie outside the plasma membrane based on
bioinformatic analyses, a theory supported by data for the localization of fasciclin family proteins. The genomic region
encoding AstaP was cloned and sequenced, and two introns
were detected (Fig. 4B). The calculated molecular mass of the
mature protein deduced from the cDNA, excluding the 20
amino acid residues at the N-terminus, is 21.2 kDa. The difference in molecular mass is estimated to be due to the glycosylation of AstaP (Fig. 4C). Based on measurements of protein
concentration and the concentration of the carotenoids extracted from the purified protein, a ratio of 0.02 mg of carotenoids mg1 of protein was obtained. The results suggest that
the binding ratio of the carotenoid is approximately 0.75
Plant Cell Physiol. 54(7): 1027–1040 (2013) doi:10.1093/pcp/pct080 ! The Author 2013.
1031
S. Kawasaki et al.
A
B
Fig. 5 Alignment and phylogenetic tree of the deduced sequence of AstaP with some of the homologs from the top 30 hits in the BLAST and
FASTA search results. (A) The sequences were aligned using the program CLUSTAL W (http://www.ebi.ac.uk/Tools/msa/clustalw2/), and shaded
by BoxShade (http://www.ch.embnet.org/software/BOX_form.html). Conserved residues are shaded black and similar residues are indicated in
light gray. ‘Dots’ indicate the residues of the putative fasciclin H1 and H2 domains. Red residues are the putative N-terminal hydrophobic signal
sequence deduced by the Signal-P program. Blue residues are the experimentally predicted N-terminal signal sequence in AstaP. Green residues
are the putative N-glycosylation sites. Nem_vecten, a sea anemone Nematostella vectensis (accession No. XP_001629263); Lyng_maju, a marine
cyanobacterium Lyngbya majuscula 3L (accession No. ZP_08426867); Sy_sll1483, Synechocystis sp. PCC6803 (accession No. NP_442911);
Roseo_sp, a marine Roseobacter clade bacterium HTCC2150 (accession No. ZP_01743234); Algori_sp, a marine bacterium Algoriphagus sp.
PR1 (accession No. ZP_07718665); Id_baltica, a marine bacterium Idiomarina baltica OS145 (accession No. ZP_01043951); Baci_pseud, an
alkaliphilic bacterium Bacillus pseudofirmus OF4 (accession No. YP_003428281); Sce_ac, Scenedesmus acutus (accession No. ACB06751), Chl_vari,
Chlorella variabilis (accession No. EFN56922). (B) Neighbor–Joining phylogenetic tree of the deduced sequence of AstaP with its homologs. OCP
(Slr1963) from Synechocystis sp. PCC 6803 was used as an outgroup. The bootstrap values >50 are indicated at the branch points.
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Astaxanthin binding orange carotenoid protein
Drosophila
Fasciclin I sig
gH1
H2
H1
H2
H1
ggH2
gH1
H2
GPI 652 aa
Human
b ig-h3
sig
H1
H2
H1
H2
H1
H2
H1
H2
GPI 683 aa
Mouse
OSF-2
sig
H1
H2
H1
H2
H1
H2
H1
gH2
HBD 811 aa
Altemia
CLAP
sig
H1
gH2
H1
H2
Sea urchin
sig
EBP-α/β
H1
H2
H1
H2
Arabidopsis
g
sig
H1
AtAGP8
gg
Arabidopsis gg
sig
H1
SOS5
ggg g gg
AstaP
H1
g
332 aa
344/343 aa
H2
GPI
420 aa
H2
GPI
420 aa
g H1 g H2
GPI
440 aa
H1
H1
Volvox
gg g
Algal-CAM sig H1 H2
Mycobacterium
sig
MPB70
g
g GPI
H2
193 aa
gggH1ggH2
223 aa
sig
Fig. 6 Comparison of structures among fasciclin family proteins. Circle pins indicate potential N-glycosylation sites. The predicted glycosylphosphatidylinositol (GPI) anchors, the signal peptides (sig) and heparin-binding sites (HBD) are boxed. Red boxes represent highly conserved H1
and H2 domains. The schematic structures are for Drosophila fasciclin 1 (accession No. P10674) (Zinn et al. 1988), Human big-h3 (accession No.
Q15582) (Skonier et al. 1992), a periostin homolog of mouse OSF-2 (accession No. D13664) (Takeshita et al. 1993), Altemia CLAP (accession No.
AAZ08324) (Warner et al. 2004), sea urchin EBP-a/b (accession No. BAA82956/BAA82957) (Hirate et al. 1999), Arabidopsis AtAGP8 (accession
No. O22126) (Schultz et al. 2000), Arabidopsis SOS5 (accession No. AEE78171) (Shi et al. 2003), Volvox Algal-CAM (accession No. X80416) (Huber
and Sumper 1994), Mycobacterium bovis MPB70 (accession No. BAA07403)(Terasaka et al. 1989, Carr et al. 2003) and strain Ki-4 AstaP (accession
No. AB731756). Peptide lengths (aa: amino acids) are shown on the right.
mol mol1 of purified protein (calculated from 21.2 kDa). The
amino acid sequence of AstaP does not show significant homology to other carotenoproteins (<15% identity in overlapping
regions) including cyanobacterial OCPs, crustacyanins from
crustaceans (cructacyanin chain A1, accession No. P58989;
crustacyanin chain A3, accession No. P80007) from lobster
(Keen et al. 1991a, Keen et al. 199b), glutathione S-transferase-like protein GSTP1 (accession No. CAG29357) from
human retina (Bhosale et al. 2004) or a lutein-binding protein
(accession No. AAK06408) from silkworm (Tabunoki et. al.
2002) (data not shown), and these carotenoproteins lack a
signal peptide, fasciclin-like domains and glycosylation (Jouni
and Wells 1996).
AstaP is expressed under photooxidative stress
conditions.
Northern analysis showed the gene encoding AstaP to be highly
up-regulated at the initial stages of salt stress (1 d) and dehydration (2 d) under high light conditions when the cell still
maintains a green color (Fig. 7A). Two-dimensional (2-D)
electrophoresis demonstrated that large amounts of AstaP
accumulated 6 d from the start of 0.7 M salt stress and dehydration under high light conditions, but not under low light
conditions (Fig. 7B, C). These results indicate that AstaP is
specifically expressed in response to photooxidative stress
conditions.
AstaP shows heat-stable 1O2 quenching activity
AstaP shows 1O2 quenching activity detected by spectrofluorometry using Singlet Oxygen Sensor Green (SOSG), a fluorescent
probe highly selective for 1O2 (see the Materials and Methods).
1
O2 quenching activity of 0.2 mM AstaP as the concentration of
binding carotenoids approximately corresponds to that of
1 mM NaN3 (a potent quencher of 1O2), and the activity was
stable after heat treatment of AstaP at 100 C for 1 h (Fig. 8).
Conclusion
In stress-treated strain Ki-4, AstaP is expressed as a highly basic,
dominant protein with a spectrum peak similar to that of the
solar spectrum at sea level (Figs. 2A, 7B). The concentration of
Plant Cell Physiol. 54(7): 1027–1040 (2013) doi:10.1093/pcp/pct080 ! The Author 2013.
1033
S. Kawasaki et al.
A
0.7M NaCl
w/HL
1.5
Dehydration
w/HL
1.5
rRNA
Days of
stress
B
0
1
2
3
0M NaCl w/HL 6 days
0
2
4
0.7M NaCl w/HL 6 days
97
64
45
30
21
11
M
7
pI
11
7
pI
11
M
C
0.7M NaCl
w/LL 6 days
Dehydration
w/LL 6 days
Dehydration
w/HL 6 days
Fig. 7 Expression profiles of AstaP. (A) Northern blots of total RNA
probed with the gene encoding AstaP amplified by PCR. Strain Ki-4
was cultivated under high light conditions (w/HL) and subjected to
0.7 M NaCl stress. 0, just before the start of stress; 1–3, after 1, 2 and
3 d of salt stress, respectively. Dehydration stress was applied under
high light conditions. 0, just before the start of stress; 2 and 4, after 2
and 4 d of dehydration stress, respectively. The estimated sizes (kb) are
indicated on the left. Ethidium bromide staining of the rRNA is shown
below the autoradiogram. (B) Two-dimensional electrophoresis of the
total proteins of strain Ki-4. Strain Ki-4 was cultivated under high light
conditions and subjected to 0.7 M NaCl stress for 6 d. Arrows indicate
the range of isoelectric focusing. The large boxed spot is a peptide
whose N-terminal amino acid sequence coincides with that of AstaP
without signal sequence. The pI of AstaP coincides with the theoretical value of 10.5 obtained from the deduced amino acid sequence of
the cDNA. Molecular sizes (M) are shown on the left (kDa). (C)
Comparison of AstaP expression under different stress conditions.
Strain Ki-4 was cultivated under normal growth condition (high
light), and subjected to stress treatment under various light conditions (w/HL, high light conditions; w/LL, low light conditions). The
boxed spot is a peptide whose N-terminal amino acid sequence coincides with that of AstaP.
Fig. 8 Singlet oxygen quenching activity of AstaP. 1O2 production was
detected by using a fluorescent probe SOSG. White light was irradiated for solutions containing 5 mM SOSG, 1 mM rose bengal and
varying volumes of AstaP (the basal AstaP solution was
A484 = 2.86 cm1; the concentration was estimated to be 23 mM as
the concentration of binding carotenoids) or NaN3 (10 mM) in a
total volume 2,000 ml. Plotted data represent the mean of triplicate
assays, and the error bars represent the SDs.
of AstaP, support the conclusion that AstaP is expressed as a
heat-stable bifunctional protein to provide a sunshade effect as
well as to dissipate 1O2. 1O2 is generally considered to be produced in PSII, and is unlikely to be released from cells due to its
high reactivity. The proposed localization of AstaP in the periplasmic space as well as its high reactivity toward 1O2 suggest
that the periplasmic space may be an alternative site of 1O2
generation in strain Ki-4 under extreme photooxidative stress
conditions. The enzymatic properties of AstaP are now being
investigated in our laboratory.
Although the solubility, molecular mass and expression profile of AstaP are similar to those of cyanobacterial OCP, no
significant structural relationships have been found, indicating
that these aqueous carotenoproteins have evolved individually
in prokaryotes and eukaryotes to protect single cells against
photooxidative damage. Further studies will help to elucidate
the protein structure and functional relationships of AstaP and
its homologs distributed in hydrospheric organisms, which
could lead to the discovery of novel functions for astaxanthin
and aqueous carotenoproteins in protection against extreme
photooxidative stresses.
Materials and Methods
AstaP in the supernatant after ultracentrifugation of cell-free
extracts (CFEs; A484 = 1.25 cm1) was estimated to be 10 mM as
the concentration of binding carotenoids (Fig. 2A), and the
estimated carotenoid concentration in the aqueous phase of
a cell, calculated from the water content of the cells, is about
135 mM (equivalent to approximately A484 = 17 cm1). These
data, including the prediction of the subcellular localization
1034
Bacterial strains and growth conditions
The composition of A3 medium is as follows (mg l1): KNO3
125, MgSO47H2O 75, K2HPO4 75, KH2PO4 175, NaCl 25,
CaCl2H2O 10, FeSO47H2O 1, H3BO3 3, MnSO47H2O 2.5,
ZnSO47H2O 2, CuSO45H2O 0.1, Na2MoO4 0.1, pH 6.8. For
plate culture, 1.5% agar was added. Normal growth conditions
Plant Cell Physiol. 54(7): 1027–1040 (2013) doi:10.1093/pcp/pct080 ! The Author 2013.
Astaxanthin binding orange carotenoid protein
for both solid and liquid cultures were achieved in a growth
chamber with a 16 h/8 h light/dark regime at 28 C with high
light conditions at 800–1,200 mmol photons m2 s1 (depending on the distance from the light source). Strain Ki-4 was deposited in the National Institute of Technology and Evaluation
(NITE) Biological Resources Center (NBRC) as strain number
NBRC108794. Primers A18F (50 -TAATGATCCTTCCGCAGGTT30 ) and A18R (50 -CCTGGTTGATCCTGCCAG-30 ) were used for
amplification and sequencing of 18S rRNA gene fragments
(Spears et al. 1992). Primers ITS-F (50 -GGAAGTAAAAGTCGT
AACAAGG-30 ) and ITS-R (50 -TCCTCCGCTTATTGATATGC-30 )
were used for amplification and sequencing of the ITS region
(Baldwin 1992). Chlamydomonas reinhardtii NIES 2238
( = SAG11-32a, UTEX 89, CCAP 11/32B) was used as a control
strain in this study.
anhydrase (29 kDa), ovalbumin (44 kDa), conalbumin
(75 kDa), aldolase (158 kDa) and blue dextran (2,000 kDa) in
three independent experiments. The elution of AstaP occurred
later than that of ovalbumin, indicating that the molecular
mass is <44 kDa. To test the solubility in water, 500 ml of purified AstaP dissolved in 50 mM Tris–HCl buffer, pH 7.5, was
dialyzed against 5 liters of MilliQ (Millipore) ultra-pure water
for 6 h, and this procedure was repeated three times. The water
solubility was also tested by passage through a desalting
column PD-10 (GE Healthcare) eluted with MilliQ water.
Protein concentration was determined by the Bradford
method (Bradford 1976) using a BSA (bovine serum albumin,
Sigma) calibration curve based on three independent assays.
The mean value was used for enzyme studies.
PAS staining
Scanning electron microscopy
Cells grown in liquid A3 medium were harvested at OD750 =
1.0 cm1 (mid exponential phase) by centrifugation at 1,000g
for 1 min. Fixation was in 2% glutaraldehyde for 12 h, followed
by three rinses with the same solution, and the cells were then
fixed in 1% OsO4. The samples were sequentially dehydrated in
an ethanol gradient series. The samples were dried by critical
point drying, coated with osmium vapor using an osmium
plasma coater, and observed using a scanning electron microscope (S4800, Hitachi).
Stress treatment
Dehydration stress was started by transferring a piece of algaegrowing solid agar medium (2 cm4 cm) to a new Petri dish
(Fig. 1B, C). Salt stress was started by the addition of sterilized
NaCl when growth reached OD750 = 1.0 cm1 when cultured in
a 1 liter flask (Fig. 1B, C). Experiments were performed under
high light conditions (800–1,200 mmol photons m2 s1).
Purification of AstaP
Salt stress was started when growth reached OD750 = 1.0 cm1
by the addition of 0.7 M NaCl, and the cultures were continued
for 6 d under high light conditions in a 5 liter bottle. The harvested cells were suspended in 50 mM Tris–HCl buffer, pH 7.5,
and CFEs were obtained by disruption of the cells in a French
press (140 MPa) followed by removal of the cell debris by centrifugation at 15,000g for 15 min. A red supernatant was obtained after ultracentrifugation at 100,000g for 2 h. Orange
fractions were collected by passing the supernatant through a
CM-Sepharose Fast Flow column (GE Healthcare) and a gel
filtration column (HR100, GE Healthcare), and a single elution
peak was obtained. The orange peak fractions were concentrated, and the purified enzyme was subjected to SDS–PAGE.
As standard markers, a molecular weight marker kit (Precision
Plus ProteinTM, BioRad) was used. The molecular mass of the
purified protein was determined by gel filtration chromatography through Sephacryl S-200 HR (1.660 cm, GE Healthcare)
and calibrated with molecular standards (Pharmacia) using an
HPLC photodiode array detector: RNase (13.7 kDa), carbonic
Periodic acid–Schiff (PAS) staining was performed with commercially available staining kits (Merck) according to the manufacturer’s instructions to determine protein glycosylation in the
polyacrylamide gel.
Determination of peptide sequences
The N-terminal amino acid sequence was determined by the
Edman degradation method using a peptide sequencer
(Shimadzu PPSQ30). The purified protein was digested with
trypsin (Promega), and a fragment obtained by HPLC was
subjected to N-terminal sequencing.
Isolation of RNA and construction of a cDNA
library
To prepare a cDNA library, strain Ki-4 cells subjected to salt
stress for 1–3 d under high light conditions were harvested, and
total RNA was extracted with Trizol reagent (Roche). Poly(A)+
mRNA was isolated and used to generate a full-length cDNA
library with a Smart-Infusion PCR cloning system (Clontech).
All steps involved in cDNA synthesis were performed according
to the manufacturer’s instructions.
Cloning of the cDNA and the genomic region
encoding AstaP
We first amplified the partial cDNA encoding AstaP by PCR
using the full-length cDNA library. The degenerated sense
primer for the N-terminal sequence of AstaP (Ast5F1:
50 -GCNACNACNGCNAARCCNGC-30 , where N is A or T or C
or G, R is A or G) and a reverse primer (Infu30 : 50 -CGGGGTACG
ATGAGACACCA-30 ) were used for the first PCR. The primer
Ast5F1 was designed for the amino acid sequence ATTAKPA.
The PCR product was used for the second nested PCR using
Ast5F2: 50 -ACNWSNACNCCNGTNTAYGC-30 , where N is A or
T or C or G, W is A or T, S is G or C, Y is T or C) and the Infu30
primer. The primer Ast5F2 was designed for the amino acid
sequence TSTPVYA. The second PCR product was sequenced.
50 -RACE was performed using a SMART RACE cDNA
Amplification kit (Clontech) according to the manufacturer’s
Plant Cell Physiol. 54(7): 1027–1040 (2013) doi:10.1093/pcp/pct080 ! The Author 2013.
1035
S. Kawasaki et al.
instructions. The 30 end of the cDNA was amplified using a
gene-specific primer Ast600F (50 -AATGTATTGTGAGCGTCC
AG-30 ) and the Infu-30 primer. The full-length cDNA sequence
was obtained by juxtaposing sequences of the 50 and 30 portions. The genomic region encoding AstaP was obtained using
specific primers from the cDNA. Typical splicing motifs were
found at the 50 (GT) and 30 (AG) ends.
the binding carotenoids of AstaP were estimated by measuring
the water content of cells (wet weight – dry weight)/wet
weight100 (%) in four independent assays. Wet cells (the
cell pellet after centrifugation was lightly dried with filter
paper to remove excess liquid medium) were weighed, and
then dried in an oven at 80 C for 24 h. The water content of
the cells stressed by 0.7 M salt with high light for 6 d was estimated to be 72.9 ± 2.5% (mean ± SD).
Bioinformatics
Database searches for sequence homology were performed
using the programs blastp (http://www.ncbi.nlm.nih.gov/
BLAST/) and FASTA (http://www.genome.jp/tools/fasta/) set
to standard parameters. The putative cell localization of
AstaP was predicted using PSORT (http://psort.hgc.jp/form.
html), SignalP (http://www.cbs.dtu.dk/services/SignalP/) and
TargetP (http://www.cbs.dtu.dk/services/TargetP/). The Olinked glycosylation of serine and threonine residues was predicted by the program NetOGlyc 3.1 (http://www.cbs.dtu.dk/
services/NetOGlyc/). The N-linked glycosylation site was predicted by the program NetNGlyc 1.0 (http://www.cbs.dtu.dk/
services/NetNGlyc/). The C-terminal hydrophobic GPI (glycosylphosphatidylinositol) anchor signal sequence was predicted
by both the big-PI Plant Predictor (http://mendel.imp.ac.at/gpi/
plant_server.html) and PSORT programs. The pI and molecular
mass were predicted by GENETYX-MAC software (GENETYX
Corporation).
Phylogenetic analysis of AstaP
The phylogenetic tree of AstaP was constructed using the
Neighbor–Joining method of the program ClustalX version
2.0.11 and visualized by the Njplot version 2.3 software.
Bootstrap analysis was computed with 1,000 replicates.
Pigment extraction and determination of
concentration
All solvents used in this study were of analytical grade quality.
Carotenoids were removed from the purified AstaP protein by
successive extractions with methanol : chloroform : H2O =
12 : 5 : 3 by gentle mixing in a tube. After the addition of chloroform and water (2 : 3), the organic phase was obtained, and
evaporated to dryness under argon gas. The extracted carotenoids were completely dissolved in chloroform (5% of the final
volume), and then ethanol was added to bring the final volume
to 100%. An absorption peak that originated from the binding
carotenoids of the purified AstaP shifted from 484 nm in water
to 476 nm in ethanol after extraction. The peak height of the
absorbance after extraction was A484 : A476 = 1 : 0.95 ± 0.02
(mean ± SD) in five independent experiments. The change in
peak height is believed to be due to the loss of carotenoids
through the extraction procedures. From these results, the concentration of the binding carotenoids in AstaP was estimated
using a peak absorbance of AstaP at 484 nm and the absorption
coefficient of astaxanthin (e476 = 125 mM1 cm1 in ethanol)
(Mercadante and Egeland 2004). Intracellular concentrations of
1036
Pigment determination
The binding pigment of AstaP was determined based on the
absorption spectra obtained using an HPLC photodiode array
detector, HPLC retention times and molecular masses in highresolution LC/MS analysis in comparison with those of standard
compounds. The carotenoid composition was monitored on an
HPLC Photo Diode Array system (Hitachi L-2455) using a
CAPCEL PAK C18 reversed-phase column (1504.6 mm i.d.,
5 mm particle size, Shiseido). The solvent system was a mixture
of methanol/water (80/20, v/v, solvent A) and a mixture of
acetone/methanol (1/1, v/v, solvent B). The column was
eluted with a linear gradient: 0–4 min, 75% A : 25% B to 50%
A : 50% B; 4–20 min, 50% A : 50% B to 100% B; 20–50 min, 100%
B. The column was maintained at 30 C and the flow rate was
1 ml min1. Synthetic astaxanthin [(3S,30 S)-3,30 -dihydroxy-b,bcarotene-4,40 -dione], lutein [(3R,30 R,60 R)-b,e-carotene-3,30 diol], canthaxanthin (b,b-carotene-4,40 -dione), b-carotene
(b,b-carotene), adonirubin [(3RS)-3-hydroxy-b,b-carotene4,40 -dione] and adonixanthin [(3S,30 S)-3,30 -dihydroxy-b,b-caroten-4-one] were obtained from Carotenature. Astaxanthin,
lutein, canthaxanthin and b-carotene were also obtained
from Wako, and their HPLC profiles were confirmed to be consistent with those of Carotenature. The adonirubin standard,
which showed a single peak at 477 nm with a retention time of
21.5 min, was not detected as a binding pigment. b-Carotene
was also not detected (retention time of standard was
39.1 min). An LC/MS ion-trap time-of-flight (LC-MS IT TOF)
detector (Shimadzu) was used for high-resolution LC/MS analysis. The HPLC conditions for the separation of carotenoids are
described above. Positive and negative ion mass spectra of the
column eluate were recorded in full scan mode (m/z 100–1,000
or 200–1000) with an electrospray ionization (ESI) source. The
interface voltage was set at 4.5 or –3.5 kV. Liquid nitrogen was
used as nebulizing gas at a flow rate of 1.5 l min1. Charged
droplets and heat block temperatures were both 200 C.
Solvents were LC/MS quality. The data were analyzed by
LCMS solution v3.60 software and Formula Predictor
Software (Shimadzu). The mass accuracy of the instrument
was estimated to be ± 10 p.p.m. The MS data of the astaxanthin
standard appeared to be [MH]+ and [MNa]+ at m/z 597.3914
and 619.3763, respectively, with a predicted formula of
C40H52O4 (error from the predicted masses of [MH]+ and
[MNa]+ were 0.0024 and 0.0005 Da, respectively). The MS
data for peak P1 appeared to be [MH]+ and [MNa]+ at m/z
597.3907 and 619.3788 (C40H52O4, error = 0.0031 and
Plant Cell Physiol. 54(7): 1027–1040 (2013) doi:10.1093/pcp/pct080 ! The Author 2013.
Astaxanthin binding orange carotenoid protein
0.0030 Da), respectively. Peak P2 showed [M – H2O + H]+,
[MH] +, [MNa] + and [MK] + values of 565.4089, 583.4160,
605.4008 and 621.3709 (C40H54O3, error = 0.0049, 0.0014,
0.0043 and 0.0004 Da), respectively, which coincides with
4-ketozeaxanthin (adonixanthin) (Yokoyama et al. 1994).
Peak P3 showed a [MH]– value at 567.4248 (C40H56O2,
error = 0.0040 Da). Peak P4 showed a [MH]+ value at 565.4000
(C40H52O2, error = 0.0040 Da). Based on these data, the main
carotenoid bound to AstaP was determined to be astaxanthin
with chromatographic, UV/Vis and MS properties consistent
with those of standard astaxanthin. 1H-NMR was performed for
peak F1 that was fractionated from carotenoid extracts obtained from a crude aqueous cell extracts due to the need for
a large amount of purified pigment for detecting 1H-NMR signals. Carotenoids were extracted from aqueous cell extracts
obtained after ultracentrifugation as described above, and the
dried pigments were dissolved in acetone and passed through
a CAPCELL PAK C18 (MGII) reversed-phase column
(15010 mm i.d., 5 mm particle size). Collected peak F1
(approximtely 0.05 mg) and an astaxanthin standard (approximately 5 mg) were dissolved in CDCl3, and analyzed by 1H-NMR
(Varian INOVA 600 MHz spectrometer). 1H-NMR signals of F1
show the same profile as those of standard astaxanthin and
their reported values (Misawa et al. 1995, Yokoyama et al.
1994). Chemical shifts are given in p.p.m. relative to appropriate
solvent residual signals (CDCl3, H2O) reported in the literature
(Flumer et al. 2010). The binding ratios of carotenoids in AstaP
were calculated using the peak area on HPLC of each pigment
and its own molar extinction coefficient (Marcadante and
Egeland 2004). For peak P2 (designated as carotenoid-x), the
astaxanthin molar extinction coefficient was substituted for the
calculation.
Determination of 1O2 formation by SOSG
The 50 mM Tris–HCl, pH 7.5, buffer in which the purified protein was dissolved after gel filtration column chromatography
was replaced with 5.0 mM Tris–HCl buffer, pH 7.5, by repeated
dilution steps using an enzyme concentrator Amicon Ultra
(3,000 Da cut-off; Millipore). The basal AstaP solution had an
A484 = 2.86 cm1, and a concentration of 0.65 ± 0.03 (mean ±
SD) mg protein ml1. The same buffer solution (5.0 mM Tris–
HCl buffer, pH 7.5) was used for blank assays and for enzyme
dilution to adjust the protein concentration. Measurement of
1
O2 production was detected by using a fluorescent probe
SOSG (Molecular Probes) according to the manufacturer’s instructions (SOSG product information is available at:
http://probes.invitrogen.com/media/pis/mp36002.
pdf ?id=mp36002) and in other references (Flors et al. 2006,
Price et al. 2009, Dall’Osto et al. 2012, Lin et al. 2013). Briefly,
SOSG was dissolved in methanol to make stock solution of
5 mM, and then was diluted by MilliQ ultra-pure water to
0.5 mM. The basal AstaP solution described above was used
in this experiment. Fluorescence measurements were made in
a spectrofluorometer (S3370, Soma) using excitation/emission
of 488/525 nm for a solution containing 5 mM SOSG, 1 mM rose
bengal and several concentrations of AstaP or NaN3 in air-saturated 100 mM Tris–HCl buffer, pH 7.5, at 25 C, in a total
volume 2,000 ml. The assay solution was continuously stirred
by a magnetic bar to yield a homogenous distribution of the
molecules in the cuvette. All buffers were of analytical grade
quality and were passed through a Chelex-100 ion exchange
column to remove trace metal ions. Samples were exposed to
white light (21 W, 400–650 nm, EFD21ED, TOSHIBA) with a
light intensity of 130 mmol photons m2 s1, resulting in rose
bengal photosensitized generation of 1O2. A time-dependent
increase of fluorescence after light irradiation with or without
addition of 100 ml of varying concentrations of AstaP or NaN3
solution (several concentrations of AstaP or NaN3 solution
dissolved in 5.0 mM Tris–HCl buffer, pH 7.5, were prepared
by dilution in the same buffer) was detected. Solutions without
rose bengal and treated as above, or solutions containing all
compounds but not subjected to light exposure did not show a
detectable increase of fluorescence. Heat treatment of the
AstaP solution was performed using a PCR machine and a
PCR tube. Plotted data represent the mean of triplicate
assays, and the error bars represent the SDs.
Northern hybridization
Total RNAs from cell lysates harvested at several time points
after the start of stress under high light conditions were isolated
using TRIzolÕ (Invitrogen). Northern blotting was carried out
by standard procedures described previously (Kawasaki et al.
2005). The RNA (10 mg) was subjected to electrophoresis in
1.0% agarose gels, blotted onto nylon membranes (Hybond
N+, Amersham), and the membranes were probed with the
PCR-amplified DNA fragment encoding the target region. The
identity of the amplified DNA fragment was confirmed by size
and nucleotide sequence. Following pre-hybridization of the
RNA at 60 C for 30 min, the 32P-labeled DNA probe was hybridized to RNA on the membrane at 60 C for 12 h.
Two-dimensional electrophoresis
Cells with or without dehydration and 0.7 M salt stress under
various light conditions (low light, 100; or high light, 800–
1,200 mmol photons m2 s1) were harvested and promptly
frozen in liquid nitrogen. Proteins were sedimented by acetone
extraction, and the pellet was dried in vacuo and dissolved in 2D electrophoresis sample buffer containing 8 M urea, 30 mM
dithiothreitol, 2% (v/v) Pharmalyte 3–10 (Pharmacia) and 0.5%
Triton X-100. Both isoelectrofocusing and SDS–PAGE were performed horizontally with Maltiphor II (Pharmacia) according to
a previously described method (Kawasaki et al. 2000, Kawasaki
et al. 2004). For electrophoresis, 40 mg of total protein was
loaded onto gels. For detection, the gels were silver-stained
with a Silver Staining Kit (Invitrogen). The N-terminal amino
acid sequence of the target spot was obtained using a transferred protein on a polyvinylidene fluoride (PVDF) membrane
stained with Coomassie Brilliant Blue.
Plant Cell Physiol. 54(7): 1027–1040 (2013) doi:10.1093/pcp/pct080 ! The Author 2013.
1037
S. Kawasaki et al.
Supplementary data
Supplementary data are available at PCP online.
Funding
This study was supported in part by the Ministry of Education,
Culture, Sports, Science and Technology [Grants-in-Aid for
Scientific Research (S0801025).].
Acknowledgments
We thank many colleagues for advice and discussion, especially
Dr. Y. Niimura, Dr. T. Kodama, Dr. J. Sato, Dr. K. Takeda and Dr.
S. Ito. We thank Mr. K. Ohkoshi, Mr. Y. Miyajima, Mr. T.
Mishima, Mr. S. Tago, Mr. K. Nakashima, Mr. H. Sato, Ms. T.
Takahashi, Mr. J. Hoki and Ms. T. Ninomiya for technical assistance, Dr. M.D. Ohto for valuable discussions, and Dr. Y. Yaguchi
and Dr. T. Motohashi for performing scanning electron
microscopy.
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