Light- and redox-dependent protein phosphorylation is particularly important for regulation of photosynthetic protein complexes located in the thylakoid membranes of chlo-roplasts.. Keyw
Trang 1thylakoid membrane of Arabidopsis thaliana
Julia P Vainonen1, Yumiko Sakuragi2, Simon Stael1, Mikko Tikkanen1, Yagut Allahverdiyeva1, Virpi Paakkarinen1, Eveliina Aro1, Marjaana Suorsa1, Henrik V Scheller2, Alexander V Vener3 and Eva-Mari Aro1
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 of the key mechanisms
used by all domains of life for regulation of cellular
processes, from gene expression to metabolic control
In plants, protein phosphorylation plays crucial roles
during acclimation of the photosynthetic apparatus to
changing environmental cues [1] Light- and
redox-dependent protein phosphorylation is particularly
important for regulation of photosynthetic protein
complexes located in the thylakoid membranes of
chlo-roplasts Four major protein complexes are involved
in photosynthetic light reactions: photosystem I (PSI),
photosystem II (PSII), cytochrome b6f complex, and
ATP synthase The major phosphoproteins in the
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 of the major phospho-proteins of PSII (D1, D2, CP43 and PsbH phospho-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 in the repair cycle of the 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 of Arabidopsis thaliana plants to high levels of light revealed specific phosphorylation of a 40 kDa protein in photosynthetic thylakoid membranes The protein was identified by MS as extracellular calcium-sensing receptor (CaS), previously reported to be located in the 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 in a 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; Fm, maximal fluorescence; FOX1, plasma membrane-specific ferroxidase; Fv, 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.
Trang 2Likewise, two cytochrome b6f 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 a thylakoid 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 in the 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 of thylakoid 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 in the 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 of a novel
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 in the thylakoid
membrane and not detectable in the 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 a thylakoid 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 of Arabidopsis and analyzed the light-induced changes in protein phos-phorylation by immunoblotting with phosphothreo-nine-specific antibody (Fig 1A) This analysis revealed the phosphorylation of a novel 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 of membrane 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 thylakoid phosphoprotein and its regulation by light in thylakoids (A) Thylakoids were isolated from dark-adapted (D) leaves or leaves exposed for 3 h to low (30 lmol photonÆm)2Æs)1) (LL), growth (100 lmol photonÆm)2Æs)1) (GL) or high (600 lmol photonÆm)2Æs)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 of the 40 kDa phos-phoprotein is indicated by an arrow (B) The product ion spectrum of the doubly charged peptide ion with m ⁄ z 573.8 obtained by ESI and collision-induced fragmentation The parent ion is labeled in the 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 in the 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 (H3PO4) The lower-case ‘t’ indicates a phosphorylated Thr residue (C) Immunoblot with CaS-specific antibody [for experimental settings, see (A)].
Trang 3tography (IMAC) [19] for phosphopeptide enrichment.
The enriched phosphopeptides were analyzed by liquid
chromatography (LC)-MS⁄ MS
Besides several known phosphopeptides of the
thyla-koid membranes (supplementary Table S1), the analysis
of data allowed the identification of a novel, previously
uncharacterized phosphopeptide The product ion
spec-trum of the 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 in the Arabidopsis protein sequence
database revealed that the amino acid sequence belongs
to the C-terminus of the 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 in the 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 in the 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 of the 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 of the plasma
mem-brane localization of CaS, using heterologous
expres-sion in onion epidermis cells, which unfortunately lack
chloroplasts [20] To address this apparent discrepancy,
the subcellular localization of the 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 in the thylakoid fraction but not in
the stroma fraction (Fig 2A) The purity of the
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 in the
thy-lakoid membrane, the thythy-lakoids isolated from leaves
exposed to high light were fractionated by digitonin [6] Immunoblot analysis of thylakoid 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 in the 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 in the 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
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.
Trang 4CaS, 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 in the stn8 mutant
(Fig 4A) Analysis of the 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 of the 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 in the wild-type (Figs 1A and 4B) and the stn7 mutant (Fig 4B)
The involvement of STN8 in the 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 of the CaS-originated phosphopeptide SGtKFLPSSD with m⁄ z 573.82+from the thylakoids of only the stn8 mutant These results revealed that CaS in stn8is not phosphorylated at Thr380, and suggest either that CaS is a direct target of the STN8 protein kinase or STN8 is a crucial component of the protein phosphory-lation cascade involved in CaS phosphoryphosphory-lation
Characterization of the CaS mutant lines The mutant Arabidopsis lines with T-DNA insertion in the intron region of the 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 of the 40 kDa phosphoprotein band in thylakoids isolated from knockout plants (Fig 5B) provides definite evidence
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 light of 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.
Trang 5that this band represents CaS To verify the lack of
CaStranscripts in the 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 of the CaS mutant
to photoinhibition of PSII However, no difference in
the decrease of the variable fluorescence⁄ maximal
fluo-rescence (Fv⁄ Fm) ratio during high light illumination
(1500 lmol photonÆm)2Æs)1 for 3 h) or during
subse-quent recovery at low light (30 lmol photonÆm)2Æs)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 in the 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 in the 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 in a 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 in a 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 in the 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.
Trang 6Ostreococcus 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 – a novel thylakoid phosphoprotein and a
potential substrate of the STN8 protein kinase
The CaS protein (At5g23060) described here is a newly
identified phosphoprotein in the thylakoid 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 in the plasma membrane (Fig 2A) Neither was CaS found in the proteome study of Arabidopsis 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 of the 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 of Arabidopsis 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.
Trang 7Further 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 of the exact phoshorylation site, which
corre-sponds to Thr380 in the C-terminus of the protein
Making use of two chloroplast protein kinase mutants
of STN7 and STN8, it was possible to assign CaS as a
likely substrate of the chloroplast-targeted STN8
pro-tein kinase (Fig 4A) As STN8 propro-tein 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 in the
cata-lytic domain of STN8 [19] The phosphorylation of
CaS at the easily accessible C-terminus is in accordance
with this selectivity of the 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 of CaS, 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 a thylakoid
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 in the four
thyla-koid protein complexes were not modified in CaS
knockout mutants Also, the light sensitivity of PSII,
which is regulated by a number of thylakoid proteins
[29], was unaffected in CaS knockout mutants
There-fore, the functional roles for CaS and its
phosphoryla-tion under stress condiphosphoryla-tions 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 in Arabidopsis [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 in a 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 in a phyto-tron under the following conditions: 100 lmol pho-tonsÆm)2Æs)1 light intensity, 8 h photoperiod, 23C, and relative humidity 70%
The T-DNA insertion lines of the stn7 gene (At1g68830) (SALK 073254) and the stn8 gene (At5g01920) (SALK 060869 and SALK 064913) in the 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 of the cas gene (At5g23060) (665G12 and SALK 070416) in the Columbia background were obtained from GABI-Kat [35] and Salk Institute
Trang 8collections [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 of Arabidopsis 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 NH4HCO3
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 22C 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 H2O⁄ acetonitrile (ACN) ⁄ MeOH (1 : 1 : 1) Phos-phopeptides were eluted with 4· 10 lL of 20 mm Na2HPO4 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 lL of 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 of the 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 of the 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
Fv to Fm, measured from intact leaves with a Hansatech
Trang 9Plant 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 CX Hotstart DNA polymerase (Strategene, La
Jolla, CA, USA) and the uracil-containing primers nt114
(forward: GGCTTAAUATGGCTATGGCGGAAATGG
CAACGA) and nt115 (reverse: GGTTTAAUTAAGGATC
CTTAATTAAGCCTCAGCGGGTCGGAGCTAGGAAG
GAACTT), where the underlined sequence was included for
regeneration of a 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 37C and 25 min at
25C The reaction mix was directly used to transform
Esc-herichia coliDH10B 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 in a buffer
(100 lm acetosyringon, 10 mm MgCl2, 10 mm 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 in a greenhouse for 4 weeks at
28C 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 in a
green-house Observations of sections of the 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 in the Turku Center of Biotechnology for maintenance of the MS unit
References
1 Vener AV (2007) Environmentally modulated phosphor-ylation and dynamics of proteins in photosynthetic membranes Biochim Biophys Acta 6, 449–457
2 Michel H, Griffin PR, Shabanowitz J, Hunt DF & Ben-nett J (1991) Tandem mass spectrometry identifies sites
of three post-translational modifications of spinach light-harvesting chlorophyll protein II Proteolytic cleavage, acetylation, and phosphorylation J Biol Chem
266, 17584–17591
3 Vener AV, Harms A, Sussman MR & Vierstra RD (2001) Mass spectrometric resolution of reversible pro-tein phosphorylation in photosynthetic membranes of Arabidopsis thaliana J Biol Chem 276, 6959–6966
4 Hansson M & Vener AV (2003) Identification of three previously unknown in vivo protein phosphorylation sites in thylakoid membranes of Arabidopsis thaliana Mol Cell Proteomics 2, 550–559
5 Rintamaki E, Kettunen R & Aro EM (1996) Differen-tial D1 dephosphorylation in functional and photodam-aged photosystem II centers Dephosphorylation is a prerequisite for degradation of damaged D1 J Biol Chem 271, 14870–14875
6 Baena-Gonzalez E, Barbato R & Aro EM (1999) Role
of phosphorylation in the repair cycle and oligomeric structure of photosystem II Planta 208, 196–204
7 Allen JF (1992) Protein phosphorylation in regulation
of photosynthesis Biochim Biophys Acta 1098, 275–335
8 Rintamaki E, Martinsuo P, Pursiheimo S & Aro EM (2000) Cooperative regulation of light-harvesting com-plex II phosphorylation via the plastoquinol and ferre-doxin–thioredoxin system in chloroplasts Proc Natl Acad Sci USA 97, 11644–11649
9 Wollman FA (2001) State transitions reveal the dynam-ics and flexibility of the photosynthetic apparatus EMBO J 20, 3623–3630
Trang 1010 Aro EM & Ohad I (2003) Redox regulation of
thyla-koid protein phosphorylation Antiox Redox Signal 1,
55–67
11 Tikkanen M, Piippo M, Suorsa M, Sirpio S, Mulo P,
Vainonen J, Vener AV, Allahverdiyeva Y & Aro EM
(2006) State transitions revisited – a buffering system
for dynamic low light acclimation of Arabidopsis Plant
Mol Biol 62, 779–793
12 Khrouchtchova A, Hansson M, Paakkarinen V,
Vai-nonen JP, Zhang S, Jensen PE, Scheller HV, Vener AV,
Aro EM & Haldrup A (2005) A previously found
thyla-koid membrane protein of 14 kDa (TMP14) is a novel
subunit of plant photosystem I and is designated PSI-P
FEBS Lett 579, 4808–4812
13 Hamel P, Olive J, Pierre Y, Wollman FA & de Vitry C
(2000) A new subunit of cytochrome b6f complex
undergoes reversible phosphorylation upon state
transi-tion J Biol Chem 275, 17072–17079
14 Rinalducci S, Larsen MR, Mohammed S & Zolla L
(2006) Novel protein phosphorylation site identification
in spinach stroma membranes by titanium dioxide
mi-crocolumns and tandem mass spectrometry J Proteome
Res 5, 973–982
15 Carlberg I, Hansson M, Kieselbach T, Schroder WP,
Andersson B & Vener AV (2003) A novel plant protein
undergoing light-induced phosphorylation and release
from the photosynthetic thylakoid membranes Proc
Natl Acad Sci USA 100, 757–762
16 Turkina MV, Kargul J, Blanco-Rivero A, Villarejo A,
Barber J & Vener AV (2006) Environmentally
modu-lated phosphoproteome of photosynthetic membranes
in the green alga Chlamydomonas reinhardtii Mol Cell
Proteomics 5, 1412–1425
17 Bellafiore S, Barneche F, Peltier G & Rochaix JD
(2005) State transitions and light adaptation require
chloroplast thylakoid protein kinase STN7 Nature 433,
892–895
18 Bonardi V, Pesaresi P, Becker T, Schleiff T, Wagner R,
Pfannschmidt T, Jahns P & Leister D (2005)
Photosys-tem II core phosphorylation and photosynthetic
accli-mation require two different protein kinases Nature
437, 1179–1182
19 Vainonen JP, Hansson M & Vener AV (2005) STN8
protein kinase in Arabidopsis thaliana is specific in
phos-phorylation of photosystem II core proteins J Biol
Chem 280, 33679–33686
20 Han S, Tang R, Anderson LK, Woerner TE & Pei ZM
(2003) A cell surface receptor mediates extracellular
Ca(2+) sensing in guard cells Nature 425, 196–200
21 Tang RH, Han S, Zheng H, Cook CW, Choi CS,
Woerner TE, Jackson RB & Pei ZM (2007) Coupling
diurnal cytosolic Ca2+ oscillations to the CAS–IP3
pathway in Arabidopsis Science 315, 1423–1426
22 Friso G, Giacomelli L, Ytterberg AJ, Peltier JB,
Rudel-la A, Sun Q & Wijk KJ (2004) In-depth analysis of the
thylakoid membrane proteome of Arabidopsis thaliana chloroplasts: new proteins, new functions, and a plastid proteome database Plant Cell 16, 478–499
23 Kleffmann T, Russenberger D, von Zychlinski A, Chris-topher W, Sjo¨lander K, Gruissem W & Baginsky S (2004) The Arabidopsis thaliana chloroplast proteome reveals pathway abundance and novel protein functions Curr Biol 14, 354–362
24 Peltier JB, Ytterberg AJ, Sun Q & van Wijk KJ (2004) New functions of the thylakoid membrane proteome of Arabidopsis thalianarevealed by a simple, fast, and ver-satile fractionation strategy J Biol Chem 279, 49367– 49383
25 Hammet A, Pike BL, McNees CJ, Conlan LA, Tenis N
& Heierhorst J (2003) FHA domains as phospho-threo-nine binding modules in cell signaling IUBMB Life 55, 23–27
26 Alexandersson E, Saalbach G, Larsson C & Kjellbom P (2004) Arabidopsis plasma membrane proteomics identi-fies components of transport, signal transduction and membrane trafficking Plant Cell Physiol 45, 1543–1556
27 Millar AH, Sweetlove LJ, Giege P & Leaver CJ (2001) Analysis of the Arabidopsis mitochondrial proteome Plant Physiol 127, 1711–1727
28 Piippo M, Allahverdiyeva Y, Paakkarinen V, Suoranta
UM, Battchikova N & Aro EM (2006) Chloroplast-mediated regulation of nuclear genes in Arabidopsis tha-lianain the absence of light stress Physiol Genomics 25, 142–152
29 Kanervo E, Suorsa M & Aro EM (2007) Assembly of protein complexes in plastids In Cell and Molecular Biology of Plastids(Bock R, ed), pp 283–313 Springer-Verlag, Berlin
30 Bordo D & Bork P (2002) The rhodanese⁄ Cdc25 phos-phatase superfamily Sequence–structure–function rela-tions EMBO Rep 3, 741–746
31 Azumi Y & Watanabe A (1991) Evidence for a senes-cence-associated gene induced by darkness Plant Phys-iol 95, 577–583
32 Fulgosi H, Soll J, de Faria Maraschin S, Korthout HA, Wang M & Testerink C (2002) 14-3-3 proteins and plant development Plant Mol Biol 50, 1019–1029
33 Li J, Lee G, Van Doren SR & Walker JC (2000) The FHA domain mediates phosphoprotein interactions
J Cell Sci 113, 4143–4149
34 Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen
H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana Science 301, 653– 657
35 Rosso MG, Li Y, Strizhov N, Reiss B, Dekker K & Weisshaar B (2003) An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics Plant Mol Biol 53, 247–259