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LightregulationofCaS,anovelphosphoproteinin the
thylakoid membraneofArabidopsis thaliana
Julia P. Vainonen
1
, Yumiko Sakuragi
2
, Simon Stael
1
, Mikko Tikkanen
1
, Yagut Allahverdiyeva
1
,
Virpi Paakkarinen
1
, Eveliina Aro
1
, Marjaana Suorsa
1
, Henrik V. Scheller
2
, Alexander V. Vener
3
and Eva-Mari Aro
1
1 Department of Biology, Plant Physiology and Molecular Biology, University of Turku, Finland
2 Department of Plant Biology, Faculty of Life Sciences, University of Copenhagen, Denmark
3 Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linko
¨
ping University, Sweden
Protein phosphorylation is one ofthe key mechanisms
used by all domains of life for regulationof cellular
processes, from gene expression to metabolic control.
In plants, protein phosphorylation plays crucial roles
during acclimation ofthe photosynthetic apparatus to
changing environmental cues [1]. Light- and redox-
dependent protein phosphorylation is particularly
important for regulationof photosynthetic protein
complexes located inthethylakoid membranes of chlo-
roplasts. Four major protein complexes are involved
in photosynthetic light reactions: photosystem I (PSI),
photosystem II (PSII), cytochrome b
6
f complex, and
ATP synthase. The major phosphoproteins inthe thy-
lakoid membrane belong to PSII and its light-harvest-
ing antenna II. The application of MS combined with
affinity chromatography for phosphopeptide enrich-
ment has allowed identification ofthe major phospho-
proteins of PSII (D1, D2, CP43 and PsbH proteins)
and light-harvesting antenna II [Lhcb1, Lhcb2 and the
minor CP29 (Lhcb4) proteins] [2–4]. Phosphorylation
of PSII core proteins is believed to play an important
role inthe repair cycle ofthe reaction center pro-
tein D1 and the assembly of PSII [5,6]. Reversible
phosphorylation of light-harvesting antenna II proteins
regulates state transitions, i.e. the mechanism that
ensures a balanced excitation of PSI and PSII in
changing environmental and metabolic conditions [7–
11]. Two phosphorylated proteins have also been iden-
tified in PSI, but the biological significance of their
phosphorylation still remains to be elucidated [4,12].
Keywords
high light; protein phosphorylation; STN8
kinase; stress response; thylakoid
membrane
Correspondence
E M. Aro, Department of Biology,
Plant Physiology and Molecular Biology,
University of Turku, FI-20014 Turku, Finland
Fax: +358 2 333 5549
Tel: +358 2 333 5931
E-mail: evaaro@utu.fi
(Received 18 December 2007, revised 7
February 2008, accepted 13 February 2008)
doi:10.1111/j.1742-4658.2008.06335.x
Exposure ofArabidopsisthaliana plants to high levels oflight revealed
specific phosphorylation ofa 40 kDa protein in photosynthetic thylakoid
membranes. The protein was identified by MS as extracellular calcium-
sensing receptor (CaS), previously reported to be located inthe plasma
membrane. By confocal laser scanning microscopy and subcellular fraction-
ation, it was demonstrated that CaS localizes to the chloroplasts and is
enriched in stroma thylakoids. The phosphorylation level of CaS responded
strongly to light intensity. The light-dependent thylakoid protein kinase
STN8 is required for CaS phosphorylation. The phosphorylation site was
mapped to the stroma-exposed Thr380, located ina motif for interaction
with 14-3-3 proteins and proteins with forkhead-associated domains, which
suggests the involvement of CaS in stress responses and signaling path-
ways. The knockout Arabidopsis lines revealed a significant role for CaS in
plant growth and development.
Abbreviations
ACN, acetonitrile; CaS, calcium-sensing receptor; FHA, forkhead-associated; F
m
, maximal fluorescence; FOX1, plasma membrane-specific
ferroxidase; F
v
, variable fluorescence; GFP, green fluorescent protein; IMAC, immobilized metal affinity chromatography; LC, liquid
chromatography; P-CaS, phosphorylated form of calcium-sensing receptor; PSI, photosystem I; PSII, photosystem II; YFP, yellow fluorescent
protein.
FEBS Journal 275 (2008) 1767–1777 ª 2008 The Authors Journal compilation ª 2008 FEBS 1767
Likewise, two cytochrome b
6
f complex subunits
undergo reversible phosphorylation: subunit IV,
revealed by radioactive labeling [13], and Rieske Fe–S
protein, which undergoes N-terminal phosphorylation,
identified by MS [14]. Furthermore, a recent study
has shown that athylakoid membrane-associated
protein, TSP9, is phosphorylated at multiple sites in
response to increasing light intensity, and it is thought
to play a role in plant stress acclimation and signal
transduction [15].
A specific feature of environmentally induced thyla-
koid protein phosphorylation is an almost exclusive
phosphorylation of Thr residues inthe proteins of
both plant and green algal photosynthetic membranes
[1,16]. The use of reverse genetics has allowed identifi-
cation of two light-dependent protein kinases involved
in phosphorylation ofthylakoid proteins. STN7 pro-
tein kinase is essential for phosphorylation of Lhcb1,
Lhcb2 and Lhcb4 proteins [11,17] and, thus, for state
transitions. The homologous STN8 protein kinase is
involved inthe phosphorylation of PSII core proteins
and is absolutely essential for phosphorylation of
PsbH protein of PSII at Thr4 [18,19].
Here we report the identification ofanovel phos-
phoprotein, calcium-sensing receptor (CaS), from thy-
lakoid membranes of Arabidopsis. The protein was
previously named CaS and characterized as an extra-
cellular calcium-sensing receptor localized in plasma
membrane [20,21]. Both biochemical and immunolocal-
ization studies, however, provide strong evidence that
CaS is a chloroplast protein localized inthe thylakoid
membrane and not detectable inthe plasma mem-
brane. It is shown that the CaS protein level as well as
its phosphorylation level increase in response to
increasing light intensities. The phosphorylation site is
mapped to Thr380, and is shown to be dependent on
the STN8 protein kinase. Insertional mutagenesis of
CaS resulted in reduced growth, indicating a significant
role for CaS protein in plant growth and development.
Results
Identification of CaS as athylakoid 40 kDa
phosphoprotein
In order to investigate the molecular mechanisms
involved in acclimation of plant photosynthetic
machinery to high light intensities, we isolated thyla-
koid membranes from the leaves ofArabidopsis and
analyzed the light-induced changes in protein phos-
phorylation by immunoblotting with phosphothreo-
nine-specific antibody (Fig. 1A). This analysis revealed
the phosphorylation ofanovel polypeptide with a
molecular mass of about 40 kDa whose level of phos-
phorylation strongly increased with rising irradiance.
To identify this 40 kDa phosphoprotein, thylakoids
isolated from leaves exposed to high-light treatment
were subjected to trypsin shaving [3,4]. The surface-
exposed domains ofmembrane proteins were released
and separated from the membranes by centrifugation.
The resulting complex mixture of hydrophilic peptides
was subjected to immobilized metal affinity chroma-
A
C
B
Fig. 1. Identification of CaS as a 40 kDa thylakoidphosphoprotein and its regulation by lightin thylakoids. (A) Thylakoids were isolated from
dark-adapted (D) leaves or leaves exposed for 3 h to low (30 lmol photonÆm
)2
Æs
)1
) (LL), growth (100 lmol photonÆm
)2
Æs
)1
) (GL) or high
(600 lmol photonÆm
)2
Æs
)1
) (HL) light, and proteins were separated by SDS ⁄ PAGE and immunoblotted with phosphothreonine-specific anti-
body. Chlorophyll (0.75 lg) was loaded in each well. Well-known thylakoid phosphoproteins are marked, and the position ofthe 40 kDa phos-
phoprotein is indicated by an arrow. (B) The product ion spectrum ofthe doubly charged peptide ion with m ⁄ z 573.8 obtained by ESI and
collision-induced fragmentation. The parent ion is labeled inthe spectrum along with the fragment ion at m ⁄ z 524.8 produced after the char-
acteristic neutral loss of phosphoric acid. The detected b-ions (N-terminal) and y-ions (C-terminal) are indicated inthe spectrum as well as in
the corresponding amino acid sequence. The ions marked with an asterisk indicate that the fragments underwent neutral loss of 98 Da
(H
3
PO
4
). The lower-case ‘t’ indicates a phosphorylated Thr residue. (C) Immunoblot with CaS-specific antibody [for experimental settings,
see (A)].
CaS – novelthylakoidphosphoproteinofArabidopsis J. P. Vainonen et al.
1768 FEBS Journal 275 (2008) 1767–1777 ª 2008 The Authors Journal compilation ª 2008 FEBS
tography (IMAC) [19] for phosphopeptide enrichment.
The enriched phosphopeptides were analyzed by liquid
chromatography (LC)-MS ⁄ MS.
Besides several known phosphopeptides ofthe thyla-
koid membranes (supplementary Table S1), the analysis
of data allowed the identification ofa novel, previously
uncharacterized phosphopeptide. The product ion spec-
trum ofthe corresponding doubly charged molecular
ion with m ⁄ z 573.8 is presented in Fig. 1B. The series
of b- and y-ions revealed the peptide sequence
SGtKFLPSSD, with lowercase ‘t’ indicating phoshory-
lated Thr. A search intheArabidopsis protein sequence
database revealed that the amino acid sequence belongs
to the C-terminus ofthe expressed protein At5g23060
with deduced molecular mass 41.3 kDa, previously
described as an extracellular CaS [20].
In a parallel approach, the gel region corresponding
to the 40 kDa phosphoprotein band inthe gel
(Fig. 1A) was cut out and subjected to in-gel digestion
for protein identification by LC-MS ⁄ MS. CaS,
together with 14 other proteins, was identified from
this gel band (supplementary Table S2).
CaS-specific antibody was then used to determine
whether the increased occurrence of phosphorylated
CaS under high-light conditions (Fig. 1A) was related to
an increase inthe amount of CaS per se. As shown in
Fig. 1C, the total amount of CaS protein was not drasti-
cally changed by increasing irradiance, but the phos-
phorylated form of CaS (P-CaS) clearly accumulated
under high-light conditions as compared to darkness.
Chloroplast localization of CaS
Localization ofthe CaS phosphoprotein to the thyla-
koid membrane, as discussed above, is in good agree-
ment with proteomics studies [22–24], but strongly
contrasts with a previous report ofthe plasma mem-
brane localization ofCaS, using heterologous expres-
sion in onion epidermis cells, which unfortunately lack
chloroplasts [20]. To address this apparent discrepancy,
the subcellular localization ofthe endogenous CaS in
Arabidopsis was investigated by exploiting purified
membrane fractions and immunoblotting with purified
CaS-specific antibody. CaS was not found in purified
plasma membrane, whereas it was present in intact
chloroplasts and inthethylakoid fraction but not in
the stroma fraction (Fig. 2A). The purity ofthe mem-
brane fractions was demonstrated by using plasma
membrane-specific ferroxidase (FOX1) and thylakoid
membrane-specific D1 antibodies as specific markers
(Fig. 2A).
To further dissect the distribution of CaS inthe thy-
lakoid membrane, the thylakoids isolated from leaves
exposed to high light were fractionated by digitonin
[6]. Immunoblot analysis ofthylakoid fractions
revealed the presence of CaS both in grana and in
stroma thylakoids, and its clear enrichment in the
stroma-exposed membranes (Fig. 2B).
To further investigate the contradiction between our
data and published reports showing the targeting of
fluorescent-labeled CaS to the plasma membrane
[20,21], we fused the yellow fluorescent protein (YFP)
recombinantly to the C-terminus of CaS and tran-
siently expressed this construct in Nicotiana benthami-
ana leaves. Observations by confocal laser scanning
microscopy clearly demonstrated that the CaS–YFP
fusion protein localized in chloroplasts (Fig. 3A–C). In
stark contrast, the cytosolic YFP control accumulated
YFP fluorescence signal inthe cell periphery and
nuclei (supplementary Fig. S1). These data clearly
demonstrate that CaS predominantly resides in chlo-
roplasts. Coexpression of CaS–YFP and GWD1tp–
green fluorescent protein (GFP), a chloroplast-targeted
protein used as a marker, showed perfect overlap of
the YFP and GFP signals (Fig. 3D–F), and no signal
was detected inthe cell periphery. Coexpression of
CAS–YFP and the cytosolic GFP further illustrated
the exclusive localization of CAS–YFP in chloroplasts
(supplementary Fig. S2).
Requirement of STN8 kinase for CaS
phosphorylation
To address the question of whether one of the
two light-regulated protein kinases, STN7 or STN8, is
required for the light-dependent phosphorylation of
A
PM
CaS
CaS
FOX1
D1
Chl Th
Th ST GT
S
B
Fig. 2. Localization of CaS to chloroplasts. (A) Plasma membrane
(PM), intact chloroplasts (Chl), thylakoids (Th) and soluble stroma (S)
were isolated from wild-type Arabidopsis, and proteins were sepa-
rated by SDS ⁄ PAGE and immunoblotted with CaS-, D1- and FOX1-
specific antibodies. Five micrograms (D1) or 10 lg (CaS and FOX1)
of protein was loaded in each well. (B) The thylakoids (Th) isolated
from leaves exposed to high light were fractionated to stroma-
exposed (ST) and grana-exosed (GT) membranes. The fractions were
separated by SDS ⁄ PAGE and immunoblotted with CaS-specific anti-
body. One microgram of chlorophyll was loaded in each well.
J. P. Vainonen et al. CaS – novelthylakoidphosphoproteinof Arabidopsis
FEBS Journal 275 (2008) 1767–1777 ª 2008 The Authors Journal compilation ª 2008 FEBS 1769
CaS, we isolated thylakoids from the high-light-treated
leaves of wild-type plants and two mutant lines lacking
STN7 or STN8 (stn7 and stn8, respectively). Immuno-
blot analysis of isolated thylakoids with phosphothreo-
nine-specific antibody revealed the absence of the
40 kDa CaS phosphorylation inthe stn8 mutant
(Fig. 4A). Analysis ofthe same fractions with CaS-
specific antibody revealed similar levels of CaS in all
samples. The migration of CaS in SDS ⁄ PAGE of
thylakoid proteins isolated from the stn8 mutant was
slightly faster than those ofthe wild-type and the stn7
mutant (Fig. 4B), which is typically observed when
protein phosphorylation is altered (see also Fig. 1A).
These data suggest that CaS is almost fully phosphory-
lated under high-light conditions, as the upper band
corresponding to the phosphorylated form dominated
under high-light conditions inthe wild-type (Figs 1A
and 4B) and the stn7 mutant (Fig. 4B).
The involvement of STN8 inthe phosphorylation of
CaS was further investigated by isolation of phospho-
peptides from the wild-type and the stn7 and stn8
thylakoids, and analyzing them by LC-MS ⁄ MS. The
mapping of phosphopeptides isolated from stn8 thylak-
oids in comparison to the wild-type and stn7 showed the
specific absence ofthe CaS-originated phosphopeptide
SGtKFLPSSD with m ⁄ z 573.8
2+
from the thylakoids of
only the stn8 mutant. These results revealed that CaS in
stn8 is not phosphorylated at Thr380, and suggest either
that CaS is a direct target ofthe STN8 protein kinase or
STN8 is a crucial component ofthe protein phosphory-
lation cascade involved in CaS phosphorylation.
Characterization ofthe CaS mutant lines
The mutant Arabidopsis lines with T-DNA insertion in
the intron region ofthe CaS gene were obtained from
GABI-Kat and SALK collections. Knockout plants
were identified by immunoblot analysis of isolated thy-
lakoids with CaS-specific antibody, and the D1-specific
antibody was used as a control for equal protein
loading (Fig. 5A). The specific absence ofthe 40 kDa
phosphoprotein band in thylakoids isolated from
knockout plants (Fig. 5B) provides definite evidence
ABC
DEF
Fig. 3. Chloroplast localization of CaS–YFP
in N. benthamiana. (A–C) A leaf section
expressing CaS–YFP. (A) YFP fluorescence
(excitation 514 nm; emission 545–600 nm).
(B) Chloroplast autofluorescence (emission
650–707 nm). (C) Overlay image of (A) and
(B). (D–F) A leaf section coexpressing
CaS–YFP and GWD1tp–GFP. (D) YFP
fluorescence (excitation 514 nm; emission
545–600 nm). (E) GFP fluorescence
(excitation 488 nm; emission 495–510 nm).
(F) Overlay image of (D) and (E).
A
B
Fig. 4. CaS is a substrate for STN8 protein kinase. Thylakoids were
isolated from leaves exposed to high lightof wild-type (WT) and
mutant plants lacking either STN7 (stn7) or STN8 (stn8). The pro-
teins were separated by SDS ⁄ PAGE and immunoblotted with (A)
phosphothreonine or (B) CaS-specific antibody. The positions of
thylakoid phosphoproteins are indicated. (A) 0.75 lg Chlorophyll
was loaded in each well. (B) one microgram of chlorophyll was
loaded in each well.
CaS – novelthylakoidphosphoproteinofArabidopsis J. P. Vainonen et al.
1770 FEBS Journal 275 (2008) 1767–1777 ª 2008 The Authors Journal compilation ª 2008 FEBS
that this band represents CaS. To verify the lack of
CaS transcripts inthe mutant plants, RT-PCR analysis
of mRNA from the mutant and wild-type plants was
performed (Fig. 5C). The CaS knockout plants showed
retarded growth even under normal unstressed condi-
tions (Fig. 5D), indicating its important role in plant
growth.
To obtain further insights into the mechanisms
responsible for the observed phenotype, we analyzed
the photochemical efficiency of PSII by fluorescence
measurements and the susceptibility ofthe CaS mutant
to photoinhibition of PSII. However, no difference in
the decrease ofthe variable fluorescence ⁄ maximal fluo-
rescence (F
v
⁄ F
m
) ratio during high light illumination
(1500 lmol photonÆm
)2
Æs
)1
for 3 h) or during subse-
quent recovery at low light (30 lmol photonÆm
)2
Æs
)1
for another 3 h) was observed between the wild-type
and the CaS mutant at any time point (supplementary
Fig. S3). The whole chain electron transfer activities
were also unaffected inthe CaS mutant as compared to
the wild-type (supplementary Table S3). As CaS is an
intrinsic thylakoid protein, we then tested whether the
absence of CaS exerts any effects on the composition of
the thylakoid protein complexes. To this end, an immu-
noblot analysis was performed on the contents of repre-
sentative proteins in different thylakoid protein
complexes, including the PSI and PSII core complexes,
ATP synthase, and the lumenal oxygen-evolving
complex. This analysis revealed no significant changes
in PSII, PSI and ATP synthase inthe CaS mutant as
compared to the wild-type (supplementary Fig. S4).
Sequence analysis and domain structure
The network-based tools targetp and chlorop
(http://www.cbs.dtu.dk) strongly predict the CaS pro-
tein to be targeted to chloroplasts, with the transit pep-
tide corresponding to residues 1–33 (Fig. 6A), which
gives a molecular mass of 37.8 kDa for the mature pro-
tein. This calculated mass is in accordance with the MS
identification of CaS ina gel region around 40 kDa,
together with CYP38, FNR and several other known
proteins (supplementary Table S2). The C-terminus
contains two motifs: a noncatalytic rhodanese homol-
ogy domain (amino acids 231–352), with the putative
active residue Cys309 substituted by Asp, and a motif
that is involved in interaction with 14-3-3 proteins and
proteins with the ‘forkhead-associated’ (FHA) domain.
These domains are found ina variety of signaling pro-
teins, and can bind directly to the phosphothreonine
residue [25]. The identified phosphorylation site,
Thr380, of CaS lies within this motif (Fig. 6A).
CaS appears to be a plant-specific protein. It has
homologs in Oryza sativa (gi:41352315) and Medica-
go truncatula (gi:92878521), as well as inthe green
algae Chlamydomonas reinhardtii (gi:46093489) and
A
B
C
D
Fig. 5. Phenotype revealed by the CaS knockout plants. Immunoblot analyses of thylakoids isolated from wild-type and CaS knockout plants
using CaS-specific, D1-specific (A) or phosphothreonine-specific (B) antibody. (C) Ethidium bromide-stained gel with RT-PCR products show-
ing no cas transcript in CaS knockout mutant lines and the presence of 18S rRNA in both mutant lines and the wild-type. (D) Retarded
growth revealed by CaS knockout plants 3 weeks (upper panel) and 5 weeks (lower panel) after sowing the seeds.
J. P. Vainonen et al. CaS – novelthylakoidphosphoproteinof Arabidopsis
FEBS Journal 275 (2008) 1767–1777 ª 2008 The Authors Journal compilation ª 2008 FEBS 1771
Ostreococcus tauri (gi:116059237) (Fig. 6B). No pro-
teins with significant sequence similarity to CaS were
found in cyanobacteria. According to hydropathy
analysis (tmhmm at http://www.cbs.dtu.dk and sosui
at http://www.bp.nuap.nagoya-u.ac.jp), CaS in higher
plants has one transmembrane helix (amino acids 188–
210 in Arabidopsis), whereas the green algae proteins
do not contain any transmembrane region. Alignment
of protein sequences with clustalw (Fig. 6B) showed
that phosphorylated Thr380 is conserved in homolo-
gous proteins of green algae.
Discussion
CaS – anovelthylakoidphosphoprotein and a
potential substrate ofthe STN8 protein kinase
The CaS protein (At5g23060) described here is a newly
identified phosphoproteininthethylakoid membrane
of Arabidopsis, with its expression and phosphoryla-
tion level being strongly dependent on light intensity.
Studies of CaS (At5g23060) localization performed
in onion epidermis using transient expression of a
CaS–GFP fusion protein indicated the plasma mem-
brane as the site of CaS localization [20]. However,
the onion epidermis cells lack chloroplasts, and there-
fore the plasma membrane localization is inconclu-
sive. Similarly, the use of human embryonic kidney
cells for localization of CaS to the plasma membrane
is questionable [21], as CaS is a plant-specific protein.
To resolve the differences between those results and
the present CaS localization to thylakoids, we per-
formed immunoblot analysis of purified Arabidopsis
plasma membrane with CaS-specific antibody, which
clearly showed the absence of CaS inthe plasma
membrane (Fig. 2A). Neither was CaS found in the
proteome study ofArabidopsis plasma membrane [26],
whereas the respective studies with Arabidopsis thy-
lakoids and mitochondria revealed the presence of
CaS [22–24,27]. Moreover, we constructed the C-ter-
minal YFP fusion of CaS and tested its subcellular
localization in N. benthamiana. The overlap of CaS–
YFP signal with chloroplast autofluorescence and the
chloroplast-targeted control GWD1tp–GFP confirm
chloroplast as the primary destination of CaS
(Fig. 3).
A
B
Fig. 6. Domain structure and homologous proteins of CaS. (A) Schematic representation ofthe domain structure of CaS. Polypeptide mod-
ules are indicated as follows: TP, chloroplast transit peptide; TM, transmembrane region; rhodanese-like, rhodanese homology domain; 14-3-
3, motif for interaction with 14-3-3 proteins; FHA1, motif for interaction with forkhead-associated domain 1. The phosphorylated Thr380 is
indicated by pThr. (B) Alignment ofArabidopsis CaS with the amino acid sequences of putative homologous proteins from higher plants and
green algae. The lowercase ‘t’ above the sequence indicates phosphorylated Thr380. The predicted transmembrane domain is marked by a
dashed line above the sequence.
CaS – novelthylakoidphosphoproteinofArabidopsis J. P. Vainonen et al.
1772 FEBS Journal 275 (2008) 1767–1777 ª 2008 The Authors Journal compilation ª 2008 FEBS
Further subfractionation of thylakoids isolated from
leaves exposed to high light and probing of these frac-
tions with CaS-specific antibody showed that the
majority of CaS protein is localized to the stromal thy-
lakoids (Fig. 2B).
Evidence for CaS phosphorylation is provided by the
mapping ofthe exact phoshorylation site, which corre-
sponds to Thr380 inthe C-terminus ofthe protein.
Making use of two chloroplast protein kinase mutants
of STN7 and STN8, it was possible to assign CaS as a
likely substrate ofthe chloroplast-targeted STN8 pro-
tein kinase (Fig. 4A). As STN8 protein kinase phospho-
rylates stroma-exposed Thr residues of PSII core
proteins [18,19], the C-terminus of CaS is most likely
oriented to the stroma, where it can be involved in signal
propagation from chloroplasts to other cellular com-
partments. STN8 kinase is selective for phosphorylation
of easily accessible residues, such as N-terminal threo-
nines of D1, D2, and CP43; this might be explained by
long loops limiting access to the active site inthe cata-
lytic domain of STN8 [19]. The phosphorylation of
CaS at the easily accessible C-terminus is in accordance
with this selectivity ofthe STN8.
CaS is regulated at multiple levels according
to environmental cues
The transcript level of CaS is significantly upregulated
under normal growth irradiance as compared to dark-
ness and low-light conditions [28]. Our results demon-
strate that the high-light treatment increases the
phosphorylation level ofCaS, whereas the amount of
the protein remains at the growth light level. Thus,
CaS expression, and possibly its function, is tightly
regulated by light at two levels: transcription, and
post-translational modification by phosphorylation.
Physiological functions of CaS
CaS knockout mutants show clearly reduced growth as
compared to the wild-type. As CaS is athylakoid pro-
tein, it was first assumed that it possibly regulates the
accumulation or stability of some thylakoid protein
complexes. This, however, was not the case, as the
contents of representative proteins inthe four thyla-
koid protein complexes were not modified in CaS
knockout mutants. Also, thelight sensitivity of PSII,
which is regulated by a number ofthylakoid proteins
[29], was unaffected in CaS knockout mutants. There-
fore, the functional roles for CaS and its phosphoryla-
tion under stress conditions are more likely to be
found in signaling cascades that coordinate the growth
and responses of plants to environmental cues. The
main location of CaS in stroma-exposed thylakoid
regions is in line with its possible signaling function.
The stroma-exposed C-terminal part of CaS has a
rhodanese-like protein domain (Fig. 6A). This domain,
lacking the catalytic residues in some cases, is found in
a wide variety of functionally distinct proteins in fre-
quent association with other domain structures known
to be involved in signal transduction [30], suggesting
that CaS might play a role in sensing and signaling of
environmental cues. It has been demonstrated that
rhodanese domain proteins are associated with specific
stress conditions, including the process of leaf senes-
cence inArabidopsis [31].
The C-terminus of CaS contains also a motif for
interaction with 14-3-3 proteins and FHA domains,
according to eukaryotic linear motif prediction at
http://www.expasy.org. 14-3-3 proteins are known to
function as adaptors that mediate protein–protein inter-
actions and to be involved in signal transduction and
stress responses and also in protein import into
chloroplasts [32]. FHA domain proteins are directly
involved in signal transduction, and the interaction
between the FHA domain and target proteins is strictly
dependent on phosphorylation of Thr residues of the
target proteins [25,33]. The identified phosphorylation
site of CaS at Thr380 is located within these predicted
motifs, and its phosphorylation is intricately regulated
by environmental cues. Although direct experimental
evidence for such protein–protein interactions is still
lacking, these structural features suggest a potential role
of CaS protein ina signal transduction cascade sensing
light or redox changes in chloroplasts and propagating
the signal via direct protein–protein interactions.
Experimental procedures
Plant material and growth conditions
Arabidopsis ecotype Columbia (Col-0) was used for all
other experiments except for the transient expression, which
was carried out in tobacco. Plants were grown ina phyto-
tron under the following conditions: 100 lmol pho-
tonsÆm
)2
Æs
)1
light intensity, 8 h photoperiod, 23 °C, and
relative humidity 70%.
The T-DNA insertion lines ofthe stn7 gene (At1g68830)
(SALK 073254) and the stn8 gene (At5g01920)
(SALK 060869 and SALK 064913) inthe Columbia back-
ground were obtained from the Salk Institute [34]. Plants
homozygous for the T-DNA insertion were identified on
the basis of PCR analysis [11,19].
The T-DNA insertion lines ofthe cas gene (At5g23060)
(665G12 and SALK 070416) inthe Columbia background
were obtained from GABI-Kat [35] and Salk Institute
J. P. Vainonen et al. CaS – novelthylakoidphosphoproteinof Arabidopsis
FEBS Journal 275 (2008) 1767–1777 ª 2008 The Authors Journal compilation ª 2008 FEBS 1773
collections [34]. CaS knockout plants were identified using
purified CaS-specific antibody (see below).
Extraction of RNA and RT-PCR analysis
Total RNA of frozen leaf tissues was extracted with TRIzol
(Invitrogen, Carlsbad, CA, USA). After RNase-free DNase
treatment, 1 lg of total RNA was used to synthesize cDNA
using SuperScript III reverse transcriptase (Invitrogen) in a
40 lL reaction volume. Four microliters (1 ⁄ 10) of RT prod-
uct was used for PCR amplification with CaS-specific and
18S RNA control primers. The forward and reverse primers,
respectively, for the 18S RNA were 5¢-CTGCCAGTAGT
CATATGCTTGTC-3¢ and 5¢-GTGTAGCGCGCGTGCG
GCCC-3¢. The forward and reverse primers, respectively,
for CaS were 5¢-AAATGGCAACGAAGTCTTCAC-3¢ and
5¢-CAGTCGGAGCTAGGAAGGAA-3¢.
Isolation of plasma membrane, intact
chloroplasts, stroma and thylakoids
The plasma membrane fraction ofArabidopsis was isolated
as previously described [36]. Intact chloroplasts were iso-
lated from mature Arabidopsis leaves using a two-step Per-
coll gradient [37]. The stroma fraction was obtained after
chloroplast lysis in buffer and centrifugation at 15 000 g.
Thylakoid membranes were isolated as described previously
[38], including protease inhibitor cocktail (Complete;
Roche, Mannheim, Germany). Thylakoids were subfrac-
tionated into grana, margin and stroma lamellae by using
the digitonin method as previously described [6].
SDS ⁄ PAGE and immunoblotting
The proteins were separated by SDS ⁄ PAGE with 6 m urea
and transferred to an Immobilon poly(vinylidene difluoride)
membrane (Millipore, Bedford, MA, USA). The membranes
were blocked with 5% (w ⁄ v) milk or BSA, and incubated
with protein or phosphothreonine-specific antibody (poly-
clonal; New England Biolabs, Beverly, MA, USA). The
amount of chloroplasts loaded in gels was tested for each
antibody to give a linear response, and was varied between
0.5 and 5 lg of chloroplasts, depending on the antibody.
The MicroLink Protein Coupling kit (Pierce, Rockford, IL,
USA) was used for purification of CaS-specific antibody,
raised against the full-length protein, kindly provided by
Z. M. Pei (Duke University, Durham, NC, USA).
Phosphopeptide isolation
Isolated thylakoids were resuspended in 25 mm NH
4
HCO
3
and 10 mm NaF to a final concentration of 3 mg of
chloroplastsÆmL
)1
and incubated with MS-grade trypsin
(Promega, Madison, WI, USA) (5 lg enzyme ⁄ mg chloro-
plasts) for 3 h at 22 °C. The digestion products were frozen,
thawed, and centrifuged at 15 000 g. The supernatant was
collected, and the membranes were resuspended in water
and centrifuged again. The supernatants, both containing
released thylakoid peptides, were pooled and centrifuged at
100 000 g for 20 min. The peptides were then lyophilized
and methyl-esterified with 2 m methanolic HCl [39]. Phos-
phopeptides were enriched by IMAC as previously described
[19], with modifications. The sample was first loaded on the
IMAC column in 0.3% acetic acid in water; unbound
peptides were lyophilized again, and loaded on the IMAC
column in H
2
O ⁄ acetonitrile (ACN) ⁄ MeOH (1 : 1 : 1). Phos-
phopeptides were eluted with 4 · 10 lLof20mm Na
2
HPO
4
with 20% ACN, and desalted using POROS R3 (PerSeptive
Biosystems, Framingham, MA, USA).
LC-MS/MS
In-gel trypsin digestion was performed as previously
described [40]. Tandem MS was performed on an API
QSTAR (Applied Biosystems, Foster City, CA, USA)
equipped with a nanoelectrospray source (MDS Protana,
Odense, Denmark) and connected in-line with the nano-
HPLC system (LC Packings, Amsterdam, the Netherlands).
Eluted and dried peptide samples were dissolved in 9 lLof
2% formic acid, centrifuged for 10 min at 12 000 g, and
transferred to an autosampler vial. Aliquots (8 lL) of sam-
ples were loaded onto a C18 PepMap, 5 lm, 1 mm · 300 lm
internal diameter nano-precolumn (LC Packing), desalted
for 1.5 min, and subjected to reverse-phase chromatography
on a C18 PepMap, 3 lm, 15 cm · 75 lm internal diameter
nanoscale LC column (LC Packing). A gradient of 5–50%
ACN in 0.1% formic acid was applied for 50 min with the
flow rate of 0.2 lLÆmin
)1
. The acquisition of MS ⁄ MS data
was performed on-line using the fully automated IDA fea-
ture ofthe analyst qs software (Applied Biosystems). The
acquisition parameters were 1 s for TOF MS survey scans
and 2–3 s for the product ion scans of two most intensive
doubly or triply charged peptides. The major trypsin pep-
tides were excluded from MS ⁄ MS acquisition. Analyses of
MS ⁄ MS data were performed with the analyst qs software,
and this was followed by protein identification by mascot
with search parameters allowing for carbamidomethylation
of Cys, one miscleavage of trypsin, oxidation of Met, and
200 p.p.m. mass accuracy. mascot search parameters in the
case of phosphopeptide analysis allowed one miscleavage of
trypsin, methylation ofthe C-terminus, Asp and Glut, and
phosphorylation of Ser and Thr.
Fluorescence measurements at room
temperature
PSII photochemical efficiency was determined as a ratio of
F
v
to F
m
, measured from intact leaves with a Hansatech
CaS – novelthylakoidphosphoproteinofArabidopsis J. P. Vainonen et al.
1774 FEBS Journal 275 (2008) 1767–1777 ª 2008 The Authors Journal compilation ª 2008 FEBS
Plant Efficiency Analyser (Hansatech Instruments, King’s
Lynn, UK) after a dark incubation for 30 min.
Construction of fluorescent protein fusions
The C-terminal YFP fusion of CaS was constructed by
using a two-step USER cloning technique [41]. The CaS
coding sequence (AY341888) was amplified by PCR using
PfuTurbo C
X
Hotstart DNA polymerase (Strategene, La
Jolla, CA, USA) and the uracil-containing primers nt114
(forward: GGCTTAAUATGGCTATGGCGGAAATGG
CAACGA) and nt115 (reverse: GGTTTAAU
TAAGGATC
CTTAATTAAGCCTCAGCGGGTCGGAGCTAGGAAG
GAACTT), where the underlined sequence was included for
regeneration ofa USER cloning cassette. The PCR product
was mixed with the PacI ⁄ Nt.BbvCI-digested plasmid
pCAMBIA330035Su and treated with USER enzyme mix
(New England Biolabs) for 35 min at 37 °C and 25 min at
25 °C. The reaction mix was directly used to transform Esc-
herichia coli DH10B chemically competent cells, the positive
clone, pCAS, was obtained, and the correct insertion was
verified by sequencing. A YFP fragment was amplified by
PCR using the uracil-containing primers nt59 (forward pri-
mer: GGCTTAAUCTGGGTAGCGGTGGAATGGTGAG
CAAGGGCGAGGAG) and nt34 (reverse primer: GGTT
TAAUTTACTTGTACAGCTCGTCCAT). The product
was mixed with the PacI ⁄ Nt.BbvCI-digested pCAS, treated
with USER enzyme mix, and used to transform E. coli
DH10B. The fusion construct, pCASYFP, was verified by
sequencing and was subsequently introduced to Agrobacte-
rium tumefaciens strain C58 pGV3850 for heterologous
expression in tobacco. GWD1tp–GFP consisted of chloro-
plast transit peptide for glucan water dikinase 1 fused to
GFP, and was used as a chloroplast marker.
Transient expression and subcellular localization
in N. benthamiana
Overnight cultures of A. tumefaciens bearing appropriate
plasmid constructs were harvested, resuspended ina buffer
(100 lm acetosyringon, 10 mm MgCl
2
,10mm Mes,
pH 5.6), and were incubated at room temperature for
2 h. The attenuance of each Agrobacterium strain
was adjusted to 0.05 at 600 nm before infiltration.
N. benthamiana was grown ina greenhouse for 4 weeks at
28 °C under 16 h of daylight and at 22 °C under 8 h of
darkness. The Agrobacterium cell suspensions were infil-
trated into leaves, and the plants were placed ina green-
house. Observations of sections ofthe infiltrated leaves
were carried out by 48 h after infiltration using a confocal
scanning laser microscope (TCS SP2; Leica Microsystems,
Wetzlar, Germany). Sequential scanning of GFP and YFP
were carried out, with excitation at 488 nm and 514 nm,
respectively, and emission at 495–510 nm and 545–600 nm,
respectively. Chloroplast autofluorescence was detected at
650–707 nm. The scan speed was 800 Hz, and a line aver-
age of 8 was used.
Acknowledgements
The work was supported by the Academy of Finland,
the Finnish Ministry of Agriculture and Forestry (the
NKJ project), the Swedish Research Council for Envi-
ronment, Agriculture and Space Planning (Formas),
the Kone Foundation, and European Union FP6
contract 021313-Glytrans. We wish to thank Professor
M. Sommarin for purified plasma membranes of Ara-
bidopsis, Dr Z. M. Pei for CaS antibody, and Dr
M. Glaring for the GDW1tp–GFP construct. We are
grateful to the proteomics unit inthe Turku Center of
Biotechnology for maintenance ofthe MS unit.
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