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Plant Physiol. (1999) 121: 61-70 Arabidopsis Mutants Lacking the 43- and 54-Kilodalton Subunits of the Chloroplast Signal Recognition Particle Have Distinct Phenotypes1
Department of Plant Biology, Carnegie Institution of Washington, 260 Panama Street, Stanford, California 94305 (P.A., D.A.C.S., M.L.P., D.H.P., N.E.H.); and Departement d'Ecophysiologie Vegetale et de Microbiologie Commissariat à l'Energie Atomique/Cadarache, F-13108 St. Paul lez Durance cedex, France (L.N.)
The chloroplast signal recognition particle (cpSRP) is a protein complex consisting of 54- and 43-kD subunits encoded by the fifty-four chloroplast, which encodes cpSRP54 (ffc), and chaos (cao) loci, respectively. Two new null alleles in the ffc locus have been identified. ffc1-1 is caused by a stop codon in exon 10, while ffc1-2 has a large DNA insertion in intron 8. ffc mutants have yellow first true leaves that subsequently become green. The reaction center proteins D1, D2, and psaA/B, as well as seven different light-harvesting chlorophyll proteins (LHCPs), were found at reduced levels in the young ffc leaves but at wild-type levels in the older leaves. The abundance of the two types of LHCP was unaffected by the mutation, while two others were increased in the absence of cpSRP54. Null mutants in the cao locus contain reduced levels of the same subset of LHCP proteins as ffc mutants, but are distinguishable in four ways: young leaves are greener, the chlorophyll a/b ratio is elevated, levels of reaction center proteins are normal, and there is no recovery in the level of LHCPs in the adult plant. The data suggest that cpSRP54 and cpSRP43 have some nonoverlapping roles and that alternative transport pathways can compensate for the absence of a functional cpSRP.
Chloroplasts contain a minimum of four pathways for targeting
proteins to the thylakoid membrane (for reviews, see Cline and Henry,
1996 One striking outcome from in vitro studies was the observation that
protein substrates exhibit a strict dependence for a particular pathway. For example, the 33-kD subunit of the oxygen-evolving complex
(OE33), plastocyanin, and PSI-F use the Sec pathway (Hulford et al.,
1994 More recently, in vitro studies have been supplemented by in vivo
analysis. Null mutations in Hcf106 (Voelker and Barkan, 1995b Mutants in the cpSRP pathway have also been isolated in Arabidopsis,
and the phenotypes are much milder than those of the maize mutants
described above (Pilgrim et al., 1998 Earlier biochemical studies established that cpSRP43 and cpSRP54 form a
complex and work together to promote the biogenesis of the major LHCP,
Lhcb1 (Schuenemann et al., 1998 Plant Growth Conditions and Transformation
Extraction of Proteins and Immunoblot Analysis For assaying cpSRP54 in fresh leaves, extracts were made by harvesting tissue directly into liquid N2 and grinding in ice-cold buffer (1 M Na2HPO4, 1 mM PMSF, 1 mM benzamidine, 5 mM -amino-n-caproic acid, 10 µg/mL leupeptin, 10 µg/mL
antipain, and 1 mM
p-hydroxymercuribenzoate) with a mortar and pestle.
Homogenate was centrifuged for 2 min at 13,000g in a
microfuge at 4°C to pellet insoluble material, and soluble extracts
were analyzed directly as described previously (Pilgrim et al., 1998 ).
For assaying Chl, the pellet was resuspended in 1 mL of extraction
buffer, and 200 µL of sample was mixed with 800 µL of acetone,
centrifuged, and assayed (Arnon, 1949). The remaining sample was
pelleted and solubilized in 8 M urea and 0.1%
(w/v) SDS. Protein concentration was determined using the bicinchoninic acid assay (Smith et al., 1985). Proteins were resolved by SDS-PAGE and either stained with Coomassie Blue or transferred to
nitrocellulose membranes, followed by the detection of proteins using
enhanced chemiluminescence, as described previously (Pilgrim et al.,
1998).
Antibodies Antisera against Arabidopsis cpSRP54 (CIW 24) was raised in rabbits (Cocalico Biologicals, Reamstown, PA) against a protein expressed in Escherichia coli containing the first 33 amino acids of the mature protein fused to residues 317 to 488 followed by GSHHHHHH. The construct used to express the antigen was made as an in-frame deletion of pNH4 (Pilgrim et al., 1998). pNH4 was digested
with EcoRV and BglII, the recessed ends were
filled in with Klenow subunit, and the plasmid was religated. An IgG
fraction was prepared from crude serum by ammonium sulfate
precipitation and chromatography on DEAE-Sephadex columns (Harlow and
Lane, 1988).
Extraction of RNA, DNA, and Hybridization Analysis RNA extractions were performed using a plant total RNA kit (RNeasy, Qiagen, Valencia, CA) as recommended by the manufacturer. Northern analysis was conducted exactly as described previously (Pilgrim et al., 1998). Arabidopsis genomic DNA was isolated as described by Murray and Thompson (1980). For Southern blots, 10 µg of
DNA was digested overnight in the appropriate enzyme in a volume of 200 µL. DNA was precipitated in alcohol, fractionated on 0.7%
(w/v) agarose gels, and blotted to nitrocellulose membranes in
20× SSC as described previously (Sambrook et al., 1989). DNA was fixed
to the membrane by UV cross-linking with a Stratalinker (Stratagene),
prehybridized in hybridization buffer (6× SSC, 1× Denhardt's
solution, 0.5% [w/v] SDS, 20 µg/mL yeast tRNA, and 0.05%
[w/v] sodium PPi) for 4 h at 65°C, and hybridized to probes (2 × 106 dpm/mL) for 16 h. Filters
were washed in 0.1× SSC/0.1% SDS for 5 min at room temperature,
followed by 60-min and 5-min washes at 65°C.
PCR and Sequencing PCR was performed in a thermal cycler (MJ Research, Waterstown, MA). Generally, 50 ng of genomic DNA was amplified in a 25-µL reaction containing 0.2 mM of each deoxynucleotide, 1.5 mM MgCl2, 0.5 unit of Taq polymerase, and 10× buffer supplied by the manufacturer (MBI Fermentas, Amherst, NY). For labeling probes by PCR, 1 ng of plasmid DNA and 0.5 µM of each primer were combined with 1 nmol of dTTP, 1 nmol of dATP, 1 nmol of dGTP, and 50 pmol of dCTP in a total volume of 7 µL. Three microliters (10 pmol) of [ -32P]dCTP (3,000 µCi/mmol, NEN) and 15 µL of Chill Out (MJ Research) were added, and the sample was placed
on ice.
Gene Isolation Texas A & M University (College Station) and Institut fur Genbiologische Forschung, Berlin bacteria artificial chromosome (BAC) filters were generously provided by Joe Ecker (University of Pennsylvania, Philadelphia). Filters were hybridized against a 5 end
probe made from digesting ffc cDNA with SacI and
BglII. The probe was labeled using a random
oligonucleotide-labeling kit according to the manufacturer's
directions (Pharmacia). BAC DNA preparations were made from the strains
containing the hybridizing BAC clones. BAC DNA was restricted with
EcoRI and analyzed on Southern blots probed with the 5
SacI-BglII fragment and a 3 end
BglII-HindIII (the HindIII site
originating from the polylinker) fragment. Clones F11D5, F8C6, F2F2,
and F22J14 yielded the appropriately sized restriction fragments. The
Ffc gene was subcloned from F22J14 in three pieces: a 5-kb
EcoRI-SacI fragment containing the promoter and
the 5 end of the coding region was subcloned into the same sites of
Puc19; a 1.9-kb XhoI-BglII fragment was subcloned
into the XhoI-BamHI sites of Bluescript SK+
(Stratagene); and a 1.4-kb BglII-EcoRI fragment
extending downstream from the coding region was subcloned into the
BamHI and EcoRI sites of Bluescript SK+. The
cloning strategy left a 434-bp gap between the two BglII
sites. The missing sequence was amplified by PCR and the PCR product was sequenced directly.
CS 3149 and CS 3153 Are Allelic, Single-Gene Mutations in ffc To determine whether any of the George Redei mutants might lack cpSRP54, we acquired all 51 lines and analyzed the level of cpSRP54 in leaf tissue by immunoblot analysis. Two pigment mutants created by x-ray mutagenesis of wild-type Arabidopsis ecotype Columbia, CS 3149 (originally scored as yellow heart), and CS 3153 (originally scored as yellow-green) (Fig. 1A, e and f) contained no detectable cpSRP54 in 50 µg of total protein (data not shown).
Northern-Blot Analysis
Isolation of the Ffc Gene To facilitate the mutational analysis of the ffc mutants, we isolated the Ffc gene by screening BAC library filters and sequenced the clone (accession no. AF092168). A map depicting the 13 exons, pertinent restriction sites, and primers is shown in Figure 3A. From PCR amplification of the yUP YAC library, we tentatively mapped ffc to chromosome 5 between the markers M447 and g4090.
Identification of the Mutation in ffc1-1 Southern blotting revealed no anomalies in the ffc1-1 allele (data not shown), further suggesting that ffc1-1 had a point mutation. To test this idea, PCR products spanning the entire ffc1-1 allele were generated and directly sequenced. A single mutation was detected at position 2,055 that changed the codon for R288, CGA, into the stop codon TGA (Fig. 3A). Conceivably, a truncated protein with a predicted molecular mass of 31 kD is expressed; however, no such protein was detected. The antibody used (CIW24) was raised against a fusion protein containing the N-terminal 33 amino acids of cpSRP54 fused to the C terminus (amino acids 317 488) (Fig. 3A). Either the truncated product is unstable or the
antiserum is not effective at recognizing the N-terminal 33 amino
acids.
Identification of the Mutation in ffc1-2 Differences between the wild-type and ffc1-2 alleles were evident from the results of Southern-blot analysis (Fig. 3B). In the first three panels of Figure 3B, lanes 1 to 24, a single blot was successively hybridized to three different probes without stripping. Newly appearing bands are marked on the blots with an arrow. Probe 2, which was generated by PCR using the primers 312 and 472, hybridized to smaller XhoI (2.6 kb versus a barely visible 12 kb) and SacI/BglII fragments (1.7 versus 2.6 kb) and larger HindIII (12 versus 10 kb) and EcoRI fragments (6.0 versus 5.9 kb) in the wild type compared with ffc1-2. This result indicated that the ffc1-2 allele is altered between the SacI-BglII sites (6422,232 bp). A simple deletion can be ruled out because hybridizing fragments are both smaller and larger in the mutant. However, the
data could be explained by a large (10 kb) DNA insertion within this region, as depicted in Figure 3A.
Null ffc Mutants Have a Yellow Heart/Virescent Phenotype; chaos Mutants Are Chlorotic Having confirmed that we isolated true null mutants in the ffc gene, we compared the ffc and chaos mutants grown under identical conditions on agar plates lacking Suc. As was seen for the transgenic cosuppression lines (Pilgrim et al., 1998), both ffc mutants produced yellow
first true leaves with green cotyledons. These leaves became green
within 1 week and all subsequent leaves were green (Fig. 1A, e and f
versus Fig. 1B, e and f). Leaves in the ffc1-1 mutant plants
exhibited a premature leaf yellowing beginning in the 4th week of
growth. As this phenotype was not observed in ffc1-2 or in
cosuppressor lines, it could not have been due exclusively to the
absence of cpSRP54.
Elevated Chl a/b Ratios Are Observed in chaos But Not in ffc Mutants All Chl b is associated with LHCP, whereas Chl a is associated with both LHCP and reaction center proteins (Thornber and Highkin, 1974). Therefore, a change in the Chl
a/b ratio reflects a change in the relative abundance of
LHCP to other Chl-containing proteins. Klimyuk et al. (1999) had
previously reported that Chl a/b ratios were elevated in
chaos plants grown under a number of different light
conditions, which is consistent with the observation that these plants
had reduced levels of LHCP relative to other Chl a-containing proteins. In contrast, cosuppressor
ffc lines had wild-type Chl a/b ratios in both
the yellow first true leaves, where Chl content was reduced by over
75%, and in the older leaves, where Chl was reduced by about 30%
(Pilgrim et al., 1998). To determine whether Chl a/b ratios
were altered in ffc null mutants, measurements were made in
10- and 24-d-old leaves of ffc1-2, chaos, Columbia, and Landsberg. Elevated Chl a/b ratios were
observed in chaos in both the young and old leaves, but were
normal in the ffc1-2 leaves (Fig.
4). These data indicated that the
composition of pigment proteins is different in the two mutant lines.
Immunoblot Analysis of LHCP in chaos and ffc1-2 The most abundant proteins of the thylakoid membrane are members of the LHCP family. LHCP proteins are grouped into 12 distinct gene families (Jansson, 1994). Four gene families encode proteins associated
with PSI (Lhca14), seven gene families encode proteins associated
with PSII (Lhcb16 and psbS), and the 12th family is comprised of
early light-inducible proteins that accumulate under periods of stress
(Jansson, 1994). Together, these proteins represent about 50% of the
total Chl and a significant fraction of the membrane protein (Green and
Salter, 1996). To determine whether the levels of individual LHCP are
affected by a mutation in cpSRP, we performed immunoblot analysis with
antibodies specific for all the LHCP families except early
light-inducible proteins. Analysis was performed on the first true
leaves harvested from 10-d-old plants and the shoots of 24-d-old
plants. At least two levels of protein were loaded for each blot to
ensure that the signal was in the linear response range, and blots were
repeated at least three times. Results from representative experiments
are shown in Figure 5. Three types of
responses were observed. In the first type, exemplified by psbS and
Lhcb4, elevated levels of protein were observed at both the 10- and
24-d time points for both mutants. In the second, exemplified by Lhca2
and Lhcb6, wild-type levels of proteins were found at both time points
for both mutants. For the third response, LHCP were reduced in
chaos plants at both 10 and 24 d but were reduced in
ffc plants only at the 10-d time point. It is noteworthy that the same subset of proteins, Lhca1, Lhca3, Lhca4, Lhcb1, Lhcb2,
Lhcb3, and Lhcb5, exhibited this response. These results suggest that
at least seven of the LHCPs are dependent on the cpSRP pathway for
targeting, while four proteins are either less dependent or independent
of cpSRP.
Reaction Center Proteins Are Affected in ffc But Not in chaos The reaction center of PSII is comprised of the D1 and D2 polypeptides. Likewise, the reaction center for PSI is comprised of psaA and psaB proteins. All four polypeptides are hydrophobic Chl proteins encoded by the chloroplast genome. In a previous investigation, reaction center proteins were not found to be affected by the chaos mutation (Klimyuk et al., 1999), whereas levels
of these proteins were reduced in transgenic plants deficient in cpSRP54 (Pilgrim et al., 1998). Levels of these proteins were re-examined in both null mutants, and the results shown in Figure 6 confirm the previous findings. Levels
of reaction center proteins were comparable or greater than wild type
for the chaos mutant. Levels of reaction center proteins
were substantially reduced in the 10-d-old ffc mutant, but
were normal or even elevated in the 24-d-old plants. Thus, reaction
center proteins are adversely affected in the 10-d-old ffc
mutant and not in chaos. These results suggest the
involvement of cpSRP54, not cpSRP43, in the biogenesis of
chloroplast-encoded proteins.
Effects on Protein Transport Machinery As was previously observed, cpSRP54 and cpSRP43 were not detected in the ffc and chaos mutants, respectively (Fig. 7). In the absence of cpSRP43, levels of cpSRP54 were somewhat reduced, suggesting that the complex stabilizes cpSRP54 or that expression of cpSRP54 is influenced by the abundance of cpSRP43. In contrast, no reduction in cpSRP43 was observed in the absence of cpSRP54. Chaperones are often found at elevated levels in tissues subjected to stress. We previously observed that both Hsp70 and ClpC increased in the ffc cosuppressor lines (Pilgrim et al., 1998). Hsp70 facilitates protein folding and plays a role in
protein translocation across membranes (Craig et al., 1990). ClpC is a
protein related to Hsp90 and is found associated with the envelope
translocon (Nielsen et al., 1997). In both null mutants the levels of
ClpC were elevated at both stages of development (Fig. 7); Hsp70 did
not appear to be significantly altered (data not shown). Another
protein found at elevated levels in the mutants was cpSecY, which forms
a protein translocation channel in the thylakoid membrane (Laidler et
al., 1995; Schuenemann et al., 1999). In both mutants, the level of SecY was elevated at both developmental stages (Fig. 7). The increases in ClpC and SecY may comprise a compensation mechanism to alleviate the
defect of functional cpSRP.
Reconstitution studies have demonstrated that cpSRP43 and cpSRP54
form a protein complex, cpSRP, that is required for the biogenesis of
Lhcb1 in vitro (Schuenemann et al., 1998
2 Present address: Mendel Biotechnology, 21375 Cabot Boulevard, Hayward, CA 94545. 3 Present address: Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143-0448. 4 Present address: Paradigm Genetics, 104 Alexander Drive, P.O. Box 14528, Research Triangle Park, NC 27709. * Corresponding author; e-mail nhoffman{at}paragen.com; fax 919-381-1234. Received March 24, 1999;
accepted May 24, 1999.
We thank Joe Ecker for providing the Arabidopsis genomic library on BAC filters, Chris Somerville for the yUP YAC library, Luc Adam for the genomic DNA isolation procedure, Andrew Staehlin, Stefan Jansson, David Simpson, Bertil Andersson, Klaas Jan van Wijk, Roberto Barbato, Ken Cline, Alice Barkan, and John Shanklin for antibodies, Courtney Riggle for excellent technical assistance, and Danja Schuenemann for critically reading the manuscript.
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  H. Rutschow, A. J. Ytterberg, G. Friso, R. Nilsson, and K. J. van Wijk Quantitative Proteomics of a Chloroplast SRP54 Sorting Mutant and Its Genetic Interactions with CLPC1 in Arabidopsis Plant Physiology, September 1, 2008; 148(1): 156 - 175. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
pH pathway; integral membrane proteins use
the chloroplast signal recognition particle (cpSRP) pathway or insert
by an apparently spontaneous mechanism where no soluble or membrane
factors have been found to be required. Only the cpSec and cpSRP
pathways have soluble factor requirements. For the Sec pathway, these
factors include cpSecA (Yuan et al., 1994
