The Evolutionarily Conserved Tetratrico Peptide Repeat
Protein Pale Yellow Green7 Is Required for Photosystem I
Accumulation in Arabidopsis and Copurifies
with the Complex1
¨ ¨
Jana Stockel, Stefan Bennewitz, Paul Hein, and Ralf Oelmuller*
¨
Institut fur Allgemeine Botanik und Pflanzenphysiologie, Friedrich Schiller University, 07747 Jena, Germany
Pale yellow green7-1 (pyg7-1) is a photosystem I (PSI)-deficient Arabidopsis (Arabidopsis thaliana) mutant. PSI subunits are
synthesized in the mutant, but do not assemble into a stable complex. In contrast, light-harvesting antenna proteins of both
photosystems accumulate in the mutant. Deletion of Pyg7 results in severely reduced growth rates, alterations in leaf
coloration, and plastid ultrastructure. Pyg7 was isolated by map-based cloning and encodes a tetratrico peptide repeat protein
with homology to Ycf37 from Synechocystis. The protein is localized in the chloroplast associated with thylakoid membranes
and copurifies with PSI. An independent pyg7 T-DNA insertion line, pyg7-2, exhibits the same phenotype. pyg7 gene expression
is light regulated. Comparison of the roles of Ycf37 in cyanobacteria and Pyg7 in higher plants suggests that the ancient protein
has altered its function during evolution. Whereas the cyanobacterial protein mediates more efficient PSI accumulation, the
higher plant protein is absolutely required for complex assembly or maintenance.
Plants are of fundamental importance to maintain 2001; Shen et al., 2002a, 2002b; Lezhneva et al., 2004;
life on earth because they supply oxygen and energy Sto ¨
¨ckel and Oelmuller, 2004). This implies a strong
during photosynthesis. The basic processes of light conservation in the organization, biogenesis, structure,
capturing are organized into two multiprotein com- and dynamics of prokaryotic and eukaryotic PSI. The
plexes, PSI and PSII. Both complexes are present in initial step in PSI biogenesis is the formation of the
prokaryotic and eukaryotic organisms and composed reaction center consisting of the heterodimer PsaA/B.
of approximately the same proteins and cofactors. The formation of this complex is thought to depend on
Ultimately, light energy is converted into a proton proper and on-time delivery of all associated cofactors
gradient across the photosynthetic membrane, which including iron, sulfur, heme, etc.
gives rise to the synthesis of ATP, and drives electron Although many structural and regulatory proteins
flow through the thylakoid membrane. Among the two for PSI are evolutionarily conserved and already pres-
photosynthetic complexes, PSI is the most conserved ent in cyanobacteria, a more detailed analysis uncov-
multiprotein complex in all photosynthetic organisms ered that some of these compounds also differ in
(Pakrasi, 1995; Chitnis, 2001; Scheller et al., 2001). The function. For instance, whereas PsaC stabilizes the
molecular mechanism of PSI biogenesis is still enig- reaction center of eukaryotic PSI and is absolutely
matic, although possible scenarios have been proposed required for its function, the cyanobacterial complex is
in numerous reviews (Pakrasi, 1995; Schwabe and also functional without PsaC (Takahashi et al., 1991; Yu
Kruip, 2000; Chitnis, 2001; Scheller et al., 2001). In et al., 1995). Differences between prokaryotic and eu-
recent years, several regulatory factors for PSI biosyn- karyotic PSI complexes become even more apparent
thesis have been identified that are conserved in pro- when the roles of individual regulatory factors are
karyotic and eukaryotic organisms (Wilde et al., 1995, considered. BtpA, for instance, a factor identified for
2001; Bartsevich and Pakrasi, 1997; Boudreau et al., prokaryote PSI biogenesis, is not present in eukaryotes
1997; Ruf et al., 1997; Mann et al., 2000; Naver et al., (Bartsevich and Pakrasi, 1997). Here, we demonstrate
that deletion of a homologous protein with regulatory
function can have different consequences for PSI
1
This work was supported by Friedrich Schiller University, Jena, assembly in prokaryotes and eukaryotes. Inactivation
Germany. of pale yellow green7 (Pyg7) in the higher plant
* Corresponding author; e-mail b7oera@hotmail.com; fax 49– Arabidopsis (Arabidopsis thaliana) completely abol-
3641–949–232.
The author responsible for distribution of materials integral to the
ishes photoautotrophic growth. In contrast, inactiva-
findings presented in this article in accordance with the policy tion of Ycf37, the homologous gene in Synechocystis,
described in the Instructions for Authors (www.plantphysiol.org) is: still allows photoautotrophic growth of the cells, al-
¨
Ralf Oelmuller (b7oera@hotmail.com). though they are severely impaired in photosynthetic
Article, publication date, and citation information can be found at activity and PSI accumulation (Wilde et al., 2001). This
www.plantphysiol.org/cgi/doi/10.1104/pp.106.078147. implies that the interplay between the regulatory
870 Plant Physiology, July 2006, Vol. 141, pp. 870–878, www.plantphysiol.org Ó 2006 American Society of Plant Biologists
Pale Yellow Green7 Is Required for Photosystem I Biogenesis
factors and PSI complex formation has changed dur-
ing evolution, presumably because the role of the
regulatory factor became more specialized or strin-
gent.
RESULTS
The recessive pyg7-1 mutant was generated by ethyl
methanesulfonate mutagenesis. When grown in soil,
homozygous mutant seedlings are lethal. After germi-
nation, they developed pyg cotyledons, but no primary
leaves. The mutant can only be propagated heterotro-
phically. Even though grown on Suc-supplemented
media, pyg7-1 plants reveal a significantly reduced
growth rate and display yellowish leaf pigmentation.
Furthermore, the leaves are thinner and almost trans-
parent compared to the wild type (Fig. 1). Under UV Figure 2. The 77 K fluorescence emission spectra for wild-type (WT) and
light, the pyg7-1 mutant exhibits a high-chlorophyll mutant pyg7-1 leaves of Arabidopsis. Spectra for wild-type leaves (solid
(Chl) fluorescence phenotype. Chl content of the mu- line) and pyg7-1 leaves (dashed line) were recorded using homogenized
tant grown under high-light conditions (100 mmol m22 leaf material. Chl was excited at 420 6 10 nm. Data were normalized to
s21) was decreased by 95% 6 1% and under low-light the PSII emission peak at 682 nm. The figure is representative for spectra
conditions (5 mE m22 s21) by 91% 6 1%, indicating a of at least five extracts from 18-d-old wild-type and pyg7-1 leaves of
minor light-dependent photosensitivity of the mutant. seedlings grown under low light (5 mmol m22 s21).
Of the two pyg7 alleles (pyg7-1 and pyg7-2) available
(see Fig. 1 and below), pyg7-1 was used for detailed
analyses. 0.8 nm in the mutant (Fig. 2). This blue shift indicates
Wild-type and pyg7-1 plants grown under axenic that the amount of the various Lhca components rela-
conditions were analyzed for P700 absorbance changes, tive to each other is changed. Western analyses revealed
77 K emission, and Chl a fluorescence. The mutant that the Lhca1, 2, and 4 proteins are only slightly
pyg7-1 did not show any detectable absorbance changes reduced in the mutant, whereas Lhca3 is reduced by
of P700 at 810 nm, indicating that PSI is not functional more than 80% (see Fig. 5B). These data are consistent
(data not shown). The 77 K fluorescence spectra were with the idea that the impairment in pyg7-1 is likely to
recorded to examine the distribution of excitation en- be caused by lesions in those PSI proteins that are
ergy in the mutant pyg7-1 and the wild type. The directly involved in electron transfer. The smaller blue
emission band at 731.1 6 0.5 nm in the wild type, shift in pyg7-1 compared to other PSI-deficient mutants
characteristic for a functional PSI, was shifted to 727.8 6 ¨
(Haldrup et al., 2000; Lezhneva et al., 2004; Stockel and
¨
Oelmuller, 2004) might be caused by the severe reduc-
tion of Lhca3 antenna protein of PSI.
No significant shift in the emission bands at 682.4 6
1.2 nm was observed, indicating that the residual
amount of PSII is functional in the mutant.
No photochemical quenching (qP) could be mea-
sured in pyg7-1 (data not shown). The Chl a ratio of
variable to maximal fluorescence parameter (Fv/Fm)
describes the maximal efficiency of PSII photochemis-
try, which correlates with the number of functional
¨
reaction centers (Oquist et al., 1992). The reduced value
of Fv/Fm in the mutant, 0.33 6 0.08 versus 0.78 6 0.03 in
the wild type, may be caused by secondary effects
because pyg7-1 does not show any detectable absor-
bance changes of P700 at 810 nm. Because of the non-
functional PSI, photosynthetic electron transport is
blocked and PSII remains the major target of excessive
light. This has also been reported for other PSI-specific
mutants as well as mutants impaired in the cytochrome
(Cyt) b6/f complex and ATP synthase (Maiwald et al.,
Figure 1. Wild-type (WT), pyg7-1, and pyg7-2 phenotypes of 18-d-old ¨ ¨
2003; Stockel and Oelmuller, 2004). In several cases, this
seedlings grown under high light (100 mmol m22 s21; A) or low light results in the down-regulation of PSII (compare with
(5 mmol m22 s21; B). ¨ ¨ ¨
Stockel and Oelmuller, 2004; P. Hein and R. Oelmuller,
Plant Physiol. Vol. 141, 2006 871
¨
Stockel et al.
unpublished data). Taken together, these data indicate
that mutation primarily affects electron transport
through PSI.
Lack of PSI in pyg7-1 is accompanied by changes in
the plastid ultrastructure (Fig. 3). In low light, only a
few lamellae and no assimilatory starch can be de-
tected in pyg7-1. In high light, the membrane structure
in the mutant plastids is less organized (data not shown).
Northern analysis of pyg7-1 plants using representa-
tive probes for nuclear and plastid-encoded PSI, PSII,
and Cyt b6/f complex subunits, as well as the large
subunit of Rubisco revealed that expression and tran-
script accumulation are not affected in the mutant (Fig.
4). Immunoblot analyses of thylakoid proteins uncov-
ered that none of the PSI subunits (PsaA, PsaC, PsaD,
Figure 4. Northern-blot analysis for psaA/B, psaC, PsaD, PsaF, and PsaL
PsaF, PsaL, and PsaH) were detectable in pyg7-1 (Fig.
as representative transcripts for PSI, psbA, PsbO for PSII, PetC (Rieske
5A). This was independent of the light intensity in iron sulfur protein of the Cyt b6/f complex), PetH (ferredoxin-NADPH-
which the seedlings were grown (data not shown). The oxidoreductase), and large subunit of Rubisco with total RNA from
significant decrease in the level of D1, the reaction 18-d-old wild-type (WT) and pyg7-1 seedlings. Lanes were loaded with
center protein of PSII, in the mutant may be caused by 15 mg (WT; pyg7-1), 7.5 mg (WT/2), or 3.75 mg (WT/4) RNA per lane.
enhanced destabilization due to increased photoinhi- Plants were grown under high light (100 mmol m22 s21).
bition in pyg7-1 plants in higher light intensities. Sub-
units PsbS and PsbO of PSII, subunit IV, and Cyt b6 of
the Cyt b6/f complex, as well as AtpB of ATP synthase,
accumulate in comparable amounts in wild type and
pyg7-1 (Fig. 5A).
In organello labeling experiments of wild-type and
pyg7-1 chloroplast proteins with 35S-Met (Fig. 6) dem-
onstrate that all major thylakoid proteins are synthe-
sized in the mutant. This indicates that accelerated
degradation, rather than a block in protein synthesis, is
responsible for the absence of PSI reaction center
polypeptides in pyg7-1.
Although PsaA and PsbA are synthesized in the mu-
tant (Fig. 6), PsaA (and other PSI subunits) were never
detectable in western studies. In contrast, PsbA accumu-
lates in a light intensity-dependent manner. Thus, the
decrease in the steady-state PsbA protein level under
high light in pyg7-1 may be caused by photoinhibition.
Because the Fv/Fm values increase in the mutant during
recovery after photodamage (data not shown), PsbA
synthesis appears to be normal in the mutant.
The gene pyg7 has been mapped on chromosome
1 using simple sequence-length polymorphism (SSLP)
and cleaved-amplified polymorphic sequence (CAPS)
markers (see ‘‘Materials and Methods’’). Finally, CAPS
markers CAT3, F19G10-VII, and the SSLP marker CIW12
(Lukowitz et al., 2000) were chosen for high-resolution
mapping of 1,338 F2 individuals deriving from back-
crosses to the ecotype Landsberg erecta. The two
markers, CAT3 and F19G10-VII, enclose the mutant
locus with 24 and four recombinations, respectively.
Appearance of the pyg7 phenotype was examined by
segregation analysis of the following progeny. The CAPS
markers F19G10-VII and m235 could localize the muta-
tion on the bacterial artificial chromosome clone T22J18
with four and seven recombination events, respectively.
Database searches and sequence analyses of amplified
Figure 3. Electron micrographs of chloroplasts from wild-type (WT) and DNA regions from pyg7-1 uncovered that the mutation
pyg7-1 leaves. Plants were grown under low light (5 mmol m22 s21). is located in At1g22700. The gene contains five exons and
872 Plant Physiol. Vol. 141, 2006
Pale Yellow Green7 Is Required for Photosystem I Biogenesis
Figure 5. Immunoblot analyses of thylakoid protein
extracts from wild-type (WT) and pyg7-1 with anti-
sera raised against the polypeptides indicated on the
right side. Fifteen micrograms (WT, pyg7), 7.5 mg
(WT/2), 5 mg (WT/4), or 2.5 mg (WT/8) of total
membrane protein was loaded per lane. Plants
were grown under high light (100 mmol m22 s21).
four introns. The mutant sequence contains a single tetratrico peptide repeat (TPR) motifs. An antiserum
G-to-A exchange at position 621 of the coding region, was raised against a Pyg7 peptide (see ‘‘Materials and
which leads to a conversion of a Trp to a stop codon of the Methods’’). It detects a polypeptide with an apparent
resulting protein in the mutant pyg7-1. Reverse tran- molecular mass of approximately 27 kD in the thyla-
scription (RT)-PCR analyses uncovered that the pyg7 koid membrane fraction of wild-type leaf extracts. No
mRNA level is low in etiolated material and increases protein was detected in the mutant. Antisera against
approximately 8-fold upon transfer of the seedlings to the reaction center protein PsaA of PSI and the reaction
light (Fig. 7). center subunit PsbA of PSII were used as a control (Fig.
The open reading frame of pyg7 encodes a polypep- 8A). The antisera also detect a polypeptide of the
tide of 296 amino acids with a predicted molecular expected molecular mass in protein extracts from
mass of 33.7 kD. Analysis of the N-terminal sequence wild-type Synechocystis cells, which was not present
using the prediction programs ChloroP (Emanuelsson in extracts from the mutant Ycf37 (data not shown; see
et al., 1999) and TargetP (Nielsen et al., 1997; below). Immunoblot analyses of soluble and mem-
Emanuelsson et al., 2000) revealed a putative plastid- brane fractions from Percoll-purified chloroplasts re-
directing transit sequence of 61 amino acids. Thus, the vealed that Pyg7 is associated with the thylakoid
predicted mature protein possesses an estimated mo- membranes of isolated chloroplasts (Fig. 8B). The lo-
lecular mass of 26.8 kD. calization of Pyg7 was further studied in a Suc gradient
Analysis of the primary sequence of Pyg7 revealed in which the two photosystems were separated after
high homology to the cyanobacterial Ycf37 from Syn- solubilization with Triton X-100 (see ‘‘Materials and
echocystis (Wilde et al., 2001) and contains three Methods’’). Figure 9 demonstrates that the highest
Plant Physiol. Vol. 141, 2006 873
¨
Stockel et al.
regulator of PSI, which represents the homolog of the
cyanobacterial Ycf37 (Wilde et al., 2001). Immunolog-
ical studies with antisera raised against a conserved
Pyg7 polypeptide from Arabidopsis revealed that the
TPR proteins Pyg7 and Ycf37 do not accumulate in the
higher plant and cyanobacterial mutants. Both orga-
nisms are impaired in PSI activity, but differ substan-
tially in that Ycf37-deficient Synechocystis cells can grow
photoautotrophically and accumulate a functional PSI
complex, whereas the higher plant mutant is lethal
and lacks PSI completely.
Chl a fluorescence measurements demonstrate that
Fv/Fm is considerably reduced in the mutant. The
increase in QA reduction further indicates that the
electron flow downstream of PSII is blocked. Determi-
nation of absorbance changes of P700 at 810 nm revealed
that PSI in pyg7-1 is not functional. Western analysis
confirmed that essential subunits of the reaction center
are either absent or severely reduced in pyg7-1 (Fig. 5)
and the corresponding knockout line, pyg7-2. Further-
more, the blue shift in the 77 K fluorescence emission
spectrum of the mutant (Fig. 2) is in agreement with the
notion that the transfer of excitation energy from
Lhca1/Lhca4 to the PSI reaction center P700 is impaired.
A similar blue shift was also reported for plants lacking
Figure 6. Pattern of 35S-radiolabeled plastid-encoded membrane pro- PsaF (Haldrup et al., 2000) and for hcf101, another PSI-
teins from 18-d-old wild-type and pyg7-1 chloroplast proteins. Label- ¨
deficient mutant (Lezhneva et al., 2004; Stockel and
ing occurred for 7.5 min and 60,000 cpm for wild-type (WT) and ¨
Oelmuller, 2004). The fluorescence data are consistent
pyg7-1 proteins were loaded on a 15% SDS-polyacrymamide gel. The with the observation that the Lhca1, 2, and 4 proteins
positions of PsaA/B (subunits A and B of PSI) and PsbA (D1 protein of are still detectable in the mutant, although they cannot
PSII reaction center) are indicated. The proteins were identified by be associated with PSI. Interestingly, Lhca3, which is
western-blot analyses. Plants were grown under high light (100 mmol located adjacent to PsaK (Jansson et al., 1996; Ben-Shem
m22 s21). et al., 2003), is severely reduced in the mutant. Finally,
loss of PSI in pyg7-1 has strong effects on the organiza-
tion of the thylakoid membrane (Fig. 3). Stacking of
amount of Pyg7 is found in fractions that contain the
PSI reaction center (represented by PsaA and Lhca1).
The very same distribution of Pyg7 in the Suc-gradient
fractions was confirmed by mass spectrometry (MS).
To confirm that the mutated gene is responsible for
the observed phenotype, we analyzed an independent
knockout line, N807379 (see ‘‘Materials and Methods’’).
Chl a fluorescence induction data of pyg7-2 resembled
that of pyg7-1. PCR and sequence analyses with the
homozygous knockout plants confirmed the informa-
tion available in the databases. No PSI activity, PsaA,
and Pyg7 proteins were detected in pyg7-2 (data not
shown; see Fig. 1).
DISCUSSION
Several regulatory proteins involved in PSI biogen-
Figure 7. Light-induced transcript accumulation of pyg7, hcf101 (14),
esis of higher plants, Chlamydomonas and Synecho-
and psaA from 12-d-old etiolated (dark) or light-grown (light, 100 mmol
cystis, have been isolated and characterized (Wilde m22 s21) Arabidopsis seedlings or etiolated seedlings that were illumi-
et al., 1995, 2001; Bartsevich and Pakrasi, 1997; nated for 4, 10, 24, or 72 h. Amplification of actin cDNA was used as an
Boudreau et al., 1997; Ruf et al., 1997; Mann et al., internal control. Relative pyg7 mRNA levels were based on three
2000; Naver et al., 2001; Shen et al., 2002a, 2002b; independent experiments. Dark, 1.0 6 0.1; 4-h light, 4.7 6 0.2; 10-h
¨ ¨
Lezhneva et al., 2004; Stockel and Oelmuller, 2004). light, 5.7 6 0.3; 24-h light, 5.8 6 0.4; 72-h light, 8.1 6 0.5; continuous
Pyg7, a TPR protein, is a novel membrane-bound light, 8.0 6 0.3.
874 Plant Physiol. Vol. 141, 2006
Pale Yellow Green7 Is Required for Photosystem I Biogenesis
the availability and/or binding of any of the cofactors
to these subunits, or the assembly of the subunits or
cofactors into a functional PSI complex. The presence
of three TPR motifs in the C-terminal part of the
protein suggests that this region might be involved in
protein-protein interactions with other PSI subunits.
Three TPR motifs are already present in the cyanobac-
terial Ycf37 protein and they exhibit the highest degree
of sequence conservation to the eukaryotic protein. It
remains to be determined whether the cyanobacterial
Ycf37 is also associated with the PSI reaction center.
Ycf3, another regulator of PSI that interacts with PsaA
Figure 8. Immunoblot analyses of Pyg7 in comparison to Hcf101,
and PsaD in Chlamydomonas (Naver et al., 2001), also
PsbA, and PsaA. A, Thylakoid membrane extracts from 18-d-old wild- contains three TPR motifs. Boudreau et al. (1997) have
type and pyg7-1 seedlings grown in high light (100 mmol m22 s21) were shown that ycf3 is only loosely associated with PSI.
analyzed with antibodies against Pyg7, PsbA, and PsaA; the soluble Thus, the role of the TRP sequences for PSI association
protein fractions were tested with Hcf101 antibodies (Stockel and
¨ needs to be analyzed in more detail.
Oelmuller, 2004). B, Percoll-purified chloroplasts from 18-d-old wild-
¨ Pyg7 is of ancient phylogenetic origin and its homo-
type seedlings (Chl) were separated into thylakoid (Thy) and stroma log, Ycf37, from Synechocystis is already required for
fractions (Str) and analyzed with antibodies against Pyg7, Hcf101, proper PSI accumulation. However, there is a remark-
PsbA, and PsaA, respectively; 20 mg of protein were loaded per lane. able difference in the function of both proteins: Cyano-
bacteria can still grow photoautotrophically in the
absence of Ycf37; also, the reduced PSI-to-PSII ratio
grana thylakoids is comparable to wild type, whereas and the higher phycobilin-to-Chl ratio suggest a func-
stroma thylakoids are barely detectable. Like other tion of Ycf37 in PSI stability or assembly (Wilde et al.,
photosynthetic mutants, pyg7-1 does not accumulate 2001). Inactivation of the higher plant Pyg7 results in
assimilatory starch (Fig. 3; Haldrup et al., 2000; Amann complete loss of PSI. A similar evolutionary shift in
¨ ¨
et al., 2004; Lezhneva et al., 2004; Stockel and Oelmuller, function has been observed for another protein pair
2004). involved in PSI accumulation, Hcf101 from higher
Inactivation of pyg7 leads to PSI deficiency and to plants (Lezhneva et al., 2004; Sto ¨
¨ckel and Oelmuller,
the inability of the mutant to grow photoautotrophi- ¨
2004) and Slr0067 from Synechocystis (T. Stockel, un-
cally. Because transcripts for representative plastid published data). This indicates that proteins that have
and nuclear-encoded subunits of the PSI reaction accessory functions in cyanobacteria develop into cru-
center are present in wild-type amounts in the mutant, cial regulators in higher plant chloroplasts. The ycf37/
it is unlikely that Pyg7 plays a role in transcription pyg7 genes provide an interesting system to investigate
and/or transcript accumulation (Fig. 4). Furthermore, this hypothesis because homologous genes are also
organello labeling experiments of thylakoid proteins present in the cyanelle of Cyanophora paradoxa, the
revealed that major subunits of the PSI reaction center plastids of Cyanidium caldarium, Porphyra purpurea,
are synthesized in pyg7-1 (Fig. 6). Because we could and Guilliardia theka. In the green algae Chlamydomonas
isolate only a limited number of plastids from homo- rheinhardtii and higher plants, the gene was transferred
zygote mutant seedlings, pulse-chase experiments to the nucleus.
gave no reasonable results. However, we could dem-
onstrate that the radiolabel disappears much more
rapidly from PsaA in the pyg7-1 plastids compared to
wild-type plastids after transfer of the isolated organ-
elles to radioactive-free medium (data not shown). The
prediction of a plastid transit peptide suggests that
Pyg7 is a chloroplast protein. This was confirmed by
cell fractionation and immunoblot analyses as well as
MS analyses of the thylakoid subfraction (see Fig. 8).
Pyg7 is a membrane-bound protein and associated
with PSI, further supporting the idea that the protein
affects the stable accumulation of the PSI complex Figure 9. Suc density gradient centrifugation of 2% Triton X-100-
solubilized PSII and PSI complexes. Prior to loading on a 5% to 28%
rather than being involved in transcriptional or post-
linear Suc gradient, the two sedimented photosystems (see ‘‘Materials
transcriptional processes (see Figs. 4, 5, and 8). West- and Methods’’) were adjusted to 1.5 mg/mL Chl and solubilized by 2%
ern analyses and MS analyses demonstrate that Pyg7 is Triton for 50 min. Suc gradients were centrifugated for 26 h at 39,000
present in substantial amounts in purified PSI frac- rpm and fractionated from top to bottom into 15 equal fractions. Equal
tions (see Fig. 9). Pyg7 might be required for the volumes of each fraction were loaded on a SDS-polyacrylamide gel and
assembly and/or stabilization of the complex. The investigated by immunoblot analyses with antibodies against PsaA,
protein can affect the stability of the PSI core subunits, Lhca1, Pyg7, and PsbA.
Plant Physiol. Vol. 141, 2006 875
¨
Stockel et al.
MATERIALS AND METHODS In Organello Radiolabeling of Proteins
Growth Conditions and Plant Material Translational active chloroplasts from 18-d-old mutant and wild-type
seedlings were purified on a Percoll gradient and resuspended in reaction
Arabidopsis (Arabidopsis thaliana) seedlings were grown in growth cham- buffer (330 mM sorbitol, 50 mM HEPES-KOH, pH 8.0, 10 mM dithiothreitol, and
bers under continuous white light and a light intensity of 5 and 100 mmol m22 100 mg/mL phenylmethylsulfonyl fluoride) according to van Wijk et al. (1995).
s21 at 22°C, respectively. For physiological experiments, seeds were sterilized For labeling, 107 chloroplasts were used. The chloroplasts were preincubated
with 33% (v/v) bleach and 0.08% N-laurylsarcrosinate, washed four times in reaction buffer containing a 5 mM amino acid mixture without Met and 10 mM
with 1 mL of sterile distilled water, and placed on petri dishes with solidified Mg-ATP for 10 min under 50 mmol m22 s21 at 23°C prior to adding 5 mCi
one-half-strength Murashige and Skoog medium (Murashige and Skoog, 35
S-Met. The reaction was stopped by adding 20 volumes of ice-cold lysis buffer
1962) supplemented with 1.35% (w/v) Suc. To ensure synchronized germi- (7 mM magnesium acetate, 118 mM potassium acetate, and 46 mM HEPES-
nation, the plates were kept in darkness at 4°C for the first 48 h. Pyg7-1 is an KOH, pH 7.6) after 7.5 min. After centrifugation at 10,000 rpm (SS34; Sorvall),
ethyl methanesulfonate-induced mutant. The T-DNA insertion line N807379, total membranes were washed twice with lysis buffer and resuspended in a
named pyg7-2, was obtained from the Nottingham Arabidopsis Stock Centre HEPES-sorbitol buffer (330 mM sorbitol, 50 mM HEPES-KOH, pH 8.0). The
(Sessions et al., 2002). Segregation and sequencing analyses using the primer radioactive incorporation of Met was measured using a scintillation counter
pairs 5#-TGTTACATAACCGGGTTGCAG-3# and 5#-TGTTTCTGCTTGAGG- (Beckman).
TTTAGATTG-3# confirmed that the pyg phenotype of the mutant is caused by
a T-DNA insertion in exon 3 (363 nucleotides downstream of the ATG codon)
of At1g22700. Isolation of Chloroplasts, Soluble Plastid Proteins,
Thylakoid Membranes, and PSI
PAM101/PDA-100 and P700 Absorbance Measurements Chloroplasts for immunolocalization analyses were isolated from 18-d-old
In vivo Chl a measurements were performed with 18-d-old Arabidop- plants. The chloroplast-enriched fraction was purified on a Percoll gradient.
sis seedlings using the pulse amplitude-modulated fluorometer PAM101, Intact chloroplasts were washed twice with isolation medium (0.3 M sorbitol,
equipped with a PAM data acquisition system (PDA-100; Walz). Prior to measure- 5 mM MgCl2, 5 mM EGTA, 5 mM Na2EDTA, 20 mM HEPES-KOH, pH 8.0, and
ments, the fiber optic of the emitter/detector unit (101-ED) was positioned 10 mM NaHCO3) and disrupted in breaking buffer (50 mM HEPES-KOH, pH
closely to the upper surface of the plants, which were dark adapted for 7 min 8.0, 10 mM MgCl2). The stromal and membrane fractions were separated by
before the minimal fluorescence F0 was recorded. A saturating white-light centrifugation at 10,000 rpm (SS34; Sorvall) for 20 min. The soluble proteins
pulse of 6,000 mmol m22 s21 for 600 ms was used to determine the maximal from the supernatant were precipitated with trichloroacetic acid and resus-
fluorescence Fm and the Fv/Fm ratio. After 1 min, actinic red light (650 nm, pended in 100 mM Na2CO3, 10% (w/v) Suc, and 50 mM dithiothreitol. The
40 mmol m22 s21) emitted from a photodiode (102 L) was turned on and the membrane proteins in the pellet were resuspended in breaking buffer.
fluorescence parameter Fm# of illuminated leaves was determined by the Separation of PSI and PSII occurred by Suc gradient centrifugation. A
application of saturating flashes every 30 s until a stable fluorescence level (Ft) crude thylakoid membrane preparation was obtained from isolated plastids.
was reached. Subsequently, the actinic light was switched off to determine the Plastids were resuspended in 10 mM Tris-HCl, pH 8.0, 5 mM KCl, 3 mM MgCl2,
minimal fluorescence F0# in the light-adapted state. The fluorescence quench- 2 mM MnCl2, and stirred on ice for 2 h. The Chl concentration was adjusted to
ing parameter qP was calculated as qP 5 (Fm# 2 Ft)/( Fm# 2 F0). The quantum 1.05 mg/mL, and the photosynthetic complexes were partially solubilized at
yield of PSII (FPSII) was calculated as FPSII 5 (Fm# 2 Ft)/Fm# and the 4°C for 50 min in the presence of 400 mM (NH4)2SO4, 30 mM octyl-b-D-
nonphotochemical quenching parameter (NPQ) as NPQ 5 (Fm 2 Fm#)/Fm#. glucopyranoside, and 0.45% sodium cholate. The photosynthetic complexes
The light-induced in vivo absorbance changes of P700 at 810 nm were were collected by high-speed centrifugation (49,000 rpm, 2 h, 4°C; Ti45 rotor;
measured using the PAM101/PDA-100 fluorometer connected to a dual- Beckman) and the pellet was resuspended in 10 mM Tris HCl, pH 8.0, 3 mM
wavelength emitter/detector unit (ED P700DW). Saturating far-red light MgCl2, 2 mM MnCl2, and 2% Triton X-100. After adjustment of the Chl
(730 nm, 15 W m22) emitted by a far-red diode (102-FR) for 1 min was concentration to 1.5 mg/mL, the two photosystems were solubilized by
applied to oxidize P700. After 30 s of far-red light, a strong white-light pulse of stirring on ice for 50 min. The suspension was clarified by centrifugation
6,000 mmol m22 s21 was applied for 400 ms. The maximal signal difference (20,000 rpm, 20 min; Sorvall) and 500-ml aliquots were loaded onto a linear Suc
(DA810max) between the reduced and the oxidized states of P700 was used to gradient (10 mL, 5%–28%; centrifugation 26 h, 39,000 rpm; SW40 rotor;
estimate the photochemical capacity of PSI (Barth and Krause, 2002). Beckman). The gradient was fractionated and aliquots of the fractions were
¨
used for MS or SDS-PAGE (Schagger and von Jagow, 1987).
77 K Measurements
Positional Cloning of pyg7-1
Fluorescence spectra at 77 K were recorded using a FluoroMax-2 fluorom-
eter (Jobin Ivon). The excitation was set at 420 6 10 nm and the spectra were A segregating F2 progeny was generated by crosses of male pollen donor
measured over 650–750 nm to reveal fluorescence emitted from PSII and PSI. plants of heterozygous lines of pyg7-1 in Columbia background with female
Leaf tissue of 18-d-old plant material was homogenized in 2 mL of reaction recipient plants of Landsberg erecta ecotype, followed by selfing of the resulting
buffer (50 mM MES-NaOH, pH 6.0, 10 mM MgCl2, 5 mM CaCl2, and 25% F1 plants. To assign the mutant locus to one of the Arabidopsis chromosomes,
glycerol) and immediately used for recording spectra. 30 F2 plants homozygous for the mutant pyg7 locus as well as a combination of
SSLPs, nga248, nga280, nga111, nga168, nga162, nga8, nga6, nga76, nga151 (Bell
and Ecker, 1994), and CAPS markers PhyB and AG (www.arabidopsis.org),
Photoinhibitory Measurements were used. For high-resolution mapping, genomic DNA from single leaves of
Mutant and wild-type Arabidopsis seedlings were illuminated with a 1,338 individual F2 plants was isolated. SSLP markers, nga248, SO392 (Bell and
photon flux density of 1,800 mmol m22 s21 for 1.5 h before transfer to 20 mmol Ecker, 1994), CIW12 (Lukowitz et al., 2000), m235, and CAPS markers CAT3
22 21
m s for subsequent recovery. Photoinhibition was assayed by calculating (www.arabidopsis.org), as well as the newly developed marker F19G10-VII (5#-
the Fv/Fm as a measure of the maximal photochemical efficiency of PSII. The AGTTGGTCCTCGAGCTCTCC-3# and 5#-GCTGCTTAAGAATGCGCAGC-
room temperature Chl fluorescence of 15-min dark-adapted plants was 3#), were used for fine-mapping procedures.
performed using the Fluorocam (Photon Systems Instruments).
RT-PCR and Northern-Blot Analyses
Antisera and Immunoblot Analyses
RNA for greening experiments was isolated from 7-d-old etiolated seed-
The Anti-Pyg7 antibodies were raised against the N-NKVARPRR- lings, 7-d-old etiolated seedlings illuminated for 4, 10, 24, or 72 h, as well as
DALKDRVK-C peptide (Eurogentec). For immunoblot analyses, a dilution green seedlings illuminated for 7 d with continuous white light of 100 mmol
of 1:500 was used. The PsbA, PsbS, Lhcb1, 2, 5, and 6 antibodies were obtained m22 s21 using TRIzol Reagent (Invitrogen) according to the manufacturer’s
¨
from Agrisera. All other antibodies have been described previously (Stockel instructions. RT-PCR reactions were performed with the RT-PCR kit (Revert-
¨
and Oelmuller, 2004). Aid first-strand cDNA synthesis kit; Fermentas) using the following primer
876 Plant Physiol. Vol. 141, 2006
Pale Yellow Green7 Is Required for Photosystem I Biogenesis
pairs 5#- AGGCTTCCACAGTTTTGGTTT-3# and 5#-CCCAAACATCTGAC- skillful technical assistance. The SAIL insertion line N807379 was obtained
TGCATTT-3# for psaA; 5#-CAAGACTCTCCTCACAGAAC-3# and 5#-CTC- from the Nottingham Arabidopsis Stock Centre. The nucleotide sequence of
TGAACCAAGAACCGTTG-3# for pyg7; 5#-GCTGATGTCTATGGTCCAA- pyg7 is identical to that of At1g22700 deposited in the EMBL database.
GTCTACC-3# and 5#-CAATTACCGCTGCTGTCAATGGCGC-3# for hcf101.
Actin2 (5#-GGTAACATTGTGCTCAGTGGTGG-3# and 5#-CTCGGCCTTGGA Received January 30, 2006; revised March 27, 2006; accepted March 27, 2006;
GATCCACATC-3#) was used as a control (Robinson et al., 1999). For the RT published May 5, 2006.
reactions, 1 mg mRNA, 0.5 mg oligo(dT) primers, as well as 2 units of Moloney
murine leukemia virus reverse transcriptase were used to synthesize cDNA.
In subsequent PCR reactions, under standard conditions, gene-specific
primers for pyg7, hcf101, psaA, as well as for actin2, with 25 and 23 cycles of
LITERATURE CITED
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Plant Cell 13: 1–17
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