Institut für Biologie III, Universität Freiburg, D-79104 Freiburg, Germany
The chloroplast genome of all higher plants
encodes, in its large single-copy region, a conserved
open reading frame of unknown function (ycf3), which
is split by two group II introns and undergoes RNA editing in monocotyledonous plants. To elucidate the
function of ycf3 we have deleted the reading frame
from the tobacco plastid genome by biolistic transformation. We show here that homoplasmic ycf3 plants
display a photosynthetically incompetent phenotype. Molecular analyses indicate that this phenotype is not
due to a defect in any of the general functions of the
plastid genetic apparatus. Instead, the mutant plants
specifically lack detectable amounts of all photosystem
I (PSI) subunits analyzed. In contrast, at least under
low light conditions, photosystem II subunits are still
present and assemble into a physiologically active complex. Faithful transcription of photosystem I genes as
well as correct mRNA processing and efficient transcript loading with ribosomes in the
ycf3 plants suggest a posttranslational cause of the PSI-defective phenotype. We therefore propose that ycf3 encodes an
essential protein for the assembly and/or stability of
functional PSI units. This study provides a first example
for the suitability of reverse genetics approaches to
complete our picture of the coding capacity of higher
plant chloroplast genomes.
THE complete sequence analysis of two chloroplast
genomes ten years ago (20, 29) marks a milestone in
plastid genetics and has had a profound influence
on our understanding of the structure and function of
plant organellar genomes. Detailed computer analyses of
the sequence data (41) allowed the identification of numerous regions potentially encoding novel proteins. In the
following years, most of these open reading frames could
be assigned to functional gene products involved in either
genetic system functions or in photosynthesis. However,
there are about 10 conserved reading frames left, the functions of which are still elusive. One of them is a reading frame
of 168 (tobacco) or 170 (maize) codons located in the large
single-copy region of higher plant chloroplast genomes
and interrupted by two group II introns. Referring to this
remarkable feature, it was initially designated IRF168 (intron-containing reading frame of 168 codons) (28), but later
renamed ycf3 (hypothetical chloroplast reading frame No. 3).
Several lines of evidence suggest that ycf3 encodes a
functional gene product. First, the reading frame is conserved in all land plant chloroplast genomes (15) and displays a high degree of DNA homology as well as putative
protein sequence homology (23). ycf3 homologues are also
present in the plastid genomes of several algae (13, 22, 35)
and in cyanobacteria (39). Second, ycf3 is actively transcribed, most probably as part of a polycistronic transcription unit, the synthesis of which initiates upstream of rps4
(see Fig. 1 A) (16, 23). Third, the ycf3 primary transcript
undergoes a series of mRNA maturation events: cleavage
into its monocistronic form, excision of two group II introns, and RNA editing at two sites in Zea mays (23). Both editing events restore conserved amino acid residues and
were shown to occur very early after transcription and independent of the other RNA processing steps (23).
Though circumstantial, all of this evidence supports the
assumption that the mature ycf3 mRNA is translated into
a functional polypeptide. However, the lack of homology
to any known gene does not allow predictions as to the
function of this putative gene product. To address this
problem directly, we have taken a reverse genetics approach to reveal the phenotype of plants deficient for ycf3.
This approach was made feasible by the development of a
technology for genetic transformation of higher plant plastids (36, 37). Over the past few years, a number of studies
have demonstrated the great value of chloroplast transformation for investigating virtually all aspects of plastid gene
expression in vivo. For example, this technology has been
successfully employed to study transcriptional and posttranscriptional regulation (1, 31, 33), RNA editing (5),
splicing (4) and DNA replication (32). In this study, we
have attempted to make use of plastid transformation to
uncover the function of the conserved chloroplast open
reading frame ycf3.
Plant Material and Growth Conditions
Sterile tobacco plants (Nicotiana tabacum) were grown on agar-solidified
MS medium (19) containing 30 g/liter sucrose. Homoplasmic transplastomic lines were rooted and propagated on the same medium. For protein
isolation and physiological measurements, transformed plants were kept
under low light conditions (0.4-0.5 W/m2) to minimize photooxidative
damage in the mutant chloroplasts.
List of Oligonucleotides
The following synthetic oligonucleotides were employed in this study:
P10 5 P11 5 P31 5 P32 5 P33 5 P34 5 P35 5 P36 5 Construction of a The region of the tobacco chloroplast genome containing the ycf3 reading
frame was excised from a SalI ptDNA clone (provided by P. Maliga, Piscataway, NJ) as a KpnI/SnaBI fragment corresponding to nucleotide positions 40,465-49,586 (29). The fragment was ligated into a Bluescript KS
vector (Stratagene, La Jolla, CA) cut with KpnI and Ecl136II, generating
plasmid pSR1. The ycf3 reading frame was subsequently deleted by digestion with ClaI and BsmBI. ClaI cuts 116 nucleotides upstream of the ycf3
start codon within the 5 Plastid Transformation and Selection of
Homoplasmic-transformed Tobacco Lines
Young leaves from sterile tobacco plants were bombarded with plasmid
pSR2-coated tungsten particles using the DuPont biolistic gun (PDS1000He;
BioRad, Hercules, CA) (12, 36). Primary spectinomycin-resistant lines
were selected on RMOP regeneration medium containing 500 mg/liter
spectinomycin dihydrochloride (37). Plastid transformants were identified
by PCR amplification according to standard protocols using the primer
pair P10 (complementary to the psbA 3
Isolation of Nucleic Acids and
Hybridization Procedures
Total plant DNA was isolated according to a rapid miniprep procedure
(8). Total cellular RNA was extracted using the TRIzol reagent (GIBCO
BRL, Paisley, Scotland). Restriction enzyme-digested DNA samples were
separated on 0.8% agarose gels and blotted onto Hybond N nylon membranes (Amersham Intl., Little Chalfont, UK) using standard protocols
(24). Total cellular RNA or polysome-bound RNA was electrophoresed
on formaldehyde-containing 0.8-1.5% agarose gels and transferred onto
Hybond N+ membranes. For hybridization, Isolation of Polysome Fractions and
Polysome-associated RNAs
Polysomes were purified as described in reference 3. Young mutant or
wild-type leaves (350 mg) from plants grown in sterile culture were
ground in liquid nitrogen and treated with 2 ml of polysome extraction
buffer (3). After removal of the insoluble material, polysomes were pelleted in a discontinuous sucrose gradient and subsequently fractionated in
an analytical (continuous) sucrose gradient (2). As a control, an EDTA-containing sample (20 mM in the resuspension buffer, 1 mM in the gradient) was prepared that causes release of ribosomes from the mRNA
chains, resulting in a uniform population of monosomes. The following
fractions were collected (from top to bottom, using SW65 ultracentrifuge tubes): (a) 150, (b) 700, (c) 750, (d) 900, and (e) 900 µl. All fractions were
diluted with 0.6 vol water before RNA isolation to reduce their sucrose
content. RNA was extracted from individual fractions by adding EDTA
(final concentration 20 mM) and phenol/chloroform (1:1 vol/vol). Subsequently, the RNA was precipitated with isopropanol after addition of 10 µg glycogen (Boehringer Mannheim). RNA pellets were resuspended in
20 µl sterile distilled water, and aliquots of 3 µl (fractions 2-5) were
loaded on denaturing agarose gels for Northern hybridization analysis.
Protein Isolation Procedures
Thylakoid proteins from wild-type and mutant tissue were isolated according to reference 14. For preparation of soluble proteins, leaf samples
were homogenized in 2 vol of extraction buffer (300 mM sucrose, 50 mM
Tris/HCl, pH 8.0, 10 mM EDTA, 2 mM EGTA, 10 mM DTT, 1 mM Pefabloc [Boehringer Mannheim] and passed through two layers of Miracloth (Calbiochem-Novabiochem, La Jolla, CA). The filtrate was centrifuged for 10 min at 15,000 g, and the supernatant was subsequently
subjected to an additional centrifugation step under identical conditions.
SDS-PAGE and Western Blot Analyses
Isolated thylakoid or soluble proteins were separated on tricine-SDS polyacrylamide gels (26) and transferred to Protran nitrocellulose BA83 membranes (Schleicher & Schuell Inc., Keene, NH) using the Trans-Blot® SD
semi-dry transfer cell (BioRad Laboratories, Hercules, CA) with a standard transfer buffer (182 mM glycine, 20 mM Tris, 20% methanol, 0.05%
SDS). Immunoblot detection was performed using the enhanced chemiluminescence system (ECL) (Amersham Intl.).
Physiological Measurements
Determination of photosystem II (PSII)1 activity was performed on
young, dark-adapted leaves from wild-type and mutant plants grown under low light conditions. PSII-dependent chlorophyll fluorescence was recorded at 650 nm with a pulsed amplitude modulation fluorimeter (Walz,
Effeltrich, Germany) (27) under illumination of intact leaf tissue with
white actinic light (flux density 10 µE/m2s; pulse frequency 100 kHz). For
complete reduction of QA, the primary quinone-type acceptor of PSII,
leaves were exposed to pulses of saturating light (700 ms; flux density
4,000 µE/m2s) every 20 s.
Deletion of the ycf3 Reading Frame from the Tobacco
Chloroplast Genome
An intron-containing reading frame of unknown function
designated ycf3 resides in the large single-copy region of
all higher plant chloroplast genomes. The position of this
open reading frame in relation to adjacent genes in the tobacco plastid DNA is depicted in Fig. 1 A. The evolutionary conservation of ycf3 as well as the existence of a cyanobacterial homologue (39) suggest an important cellular
function of this reading frame. However, the gene product
has not been identified to date, and no mutants associated with ycf3 are available. We have therefore attempted to
shed some light on the function of ycf3 by creating a null
allele and introducing it into the tobacco chloroplast genome to replace the functional copy of ycf3.
Construction of the null allele was accomplished by deleting most of the ycf3 coding region and replacing it with
a chimeric selectable marker gene (aadA) (36) in a cloned
plastid DNA fragment (Fig. 1, A and B). The transformation vector pSR2 was introduced into tobacco plastids using the biolistic protocol. Two homologous recombination
events in the flanking plastid DNA sequences result in replacement of ycf3 by aadA (Fig. 1). Since a single leaf cell in higher plants may contain up to 10,000 identical copies
of the chloroplast genome, application of high selective
pressure is required to amplify transformed plastid DNA
molecules and to eliminate wild-type genomes. This can be
achieved by regeneration of the bombarded leaf tissue under selection on spectinomycin-containing medium, since
the presence of the aadA transgene confers resistance to
aminoglycoside antibiotics (36). From the initial round of selection, we obtained several resistant lines harboring the
aadA transgene in their chloroplast genome. The primary
transformants containing a mixture of wild-type and transformed chloroplast genomes were subjected to several additional rounds of regeneration on selective medium. This
eventually resulted in mutant lines with a uniformly altered plastid DNA population. The absence of residual
wild-type genome copies was verified by DNA gel blot analysis (Fig. 2).
Complete elimination of the ycf3 reading frame results in
plants viable on sucrose-containing medium. This indicates that ycf3 is not an essential gene for plastid maintenance and plant development.
Shoots from homoplasmic
The mutant, pigment-deficient phenotype proved to be
stable under nonselective conditions, providing additional
proof for the complete absence of wild-type genome copies.
Mutant Plastids Specifically Lack Photosystem I
The phenotype of the homoplasmic transformants suggests that the ycf3 gene product is directly or indirectly involved in photosynthetic electron transfer. To test whether
the photosynthetic deficiency of the Table I.
Test for Presence of Plastid-localized Proteins in
Immunoblot analysis of soluble proteins revealed that
plastocyanin transferring the electrons from cytochrome f
to the primary PSI acceptor, P700, is also present at wild-type levels in Presence of functional PSII units in the mutant plants
was further confirmed by measurements of PSII-dependent chlorophyll fluorescence at room temperature (Fig.
5). Even a moderate light flux of as little as 80 µE/m2s
(corresponding to ~6% of normal sunlight) resulted in a
completely reduced pool of the primary quinone-type acceptor QA. This finding indicates that the electrons generated by PSII are not efficiently accepted by one of the
downstream components of the electron transfer chain.
Transcription and RNA Stability of Photosystem I
Genes Are not Impaired in Mutant Plastids
Several possible reasons for the lack of PSI protein accumulation in To exclude the possibility that the absence of ycf3 protein specifically impairs PSI gene transcription or RNA
stability, mRNA accumulation was tested for all plastid-encoded PSI genes: psaA, psaB, psaC, psaI, and psaJ.
psaA and psaB (encoding the two P700-chlorophyll a apoproteins of PSI) had to be analyzed also for a second reason: they are located downstream of ycf3, and replacement
of ycf3 with the chimeric aadA gene theoretically could exert a negative effect on psaA/B transcription.
psaC is located in the small, single-copy region of higher
plant plastid genomes. It is cotranscribed with six genes
homologous to NADPH dehydrogenase subunits as part
of the plastid ndhH operon (18). Hybridization with a
psaC-specific probe detects a complex transcript pattern
(Fig. 6 A), most likely resulting from cleavage of the polycistronic precursor transcript into numerous processing intermediates and from splicing of the intron-containing
ndhA gene. The major transcript of ~0.5 kb represents the
monocistronic psaC mRNA being one of the final maturation products (18). No differences between wild-type and
mutant plants could be detected in mRNA accumulation
or transcript pattern (Fig. 6 A) thus excluding a pretranslational defect as the reason for the lack of PsaC protein accumulation in
The psaA-specific probe detects a major RNA species of
5.2 kb (Fig. 6 B) spanning the cotranscribed psaA, psaB,
and rps14 genes (17). The same transcript is also present in
all of the Transcription of the other two plastid-encoded PSI
genes, psaI and psaJ, was also examined. Hybridization
with a psaI-specific probe detects a complex transcript pattern (Fig. 6 C), suggesting that at least part of the mRNA
population is synthesized by cotranscription with some of
the adjacent reading frames (40). The most abundant mRNA species of ~0.6 kb represents monocistronic psaI
message. The psaJ hybridization probe detects a prominent transcript of ~0.5 kb, in addition to a number of minor mRNA species of higher molecular weight (Fig. 6 D).
The major band most likely represents monocistronic psaJ
message since it is too small to also cover the downstream
ribosomal protein gene rpl33 (29). Again, no difference in
transcript pattern or mRNA accumulation could be observed between wild-type and These results suggest that translatable mRNAs of PSI
genes accumulate in mutant plants. We therefore conclude
that ycf3 is most likely not involved in any of the pretranslational steps in the expression of plastid-encoded PSI
genes.
Transcripts of Photosystem I Genes Are Efficiently
Loaded with Ribosomes in Since our Northern blot analyses suggest that no gene expression step before translation of PSI transcripts is
blocked in It has frequently been observed that unassembled subunits of PSI complexes are highly unstable (10, 25, 30).
The rather lengthy pulse-labeling experiments may thus
prevent the detection of PSI translation products in
Hybridization using a psaA-specific probe (Fig. 7 A) detects RNA species identical with the ones identified in our
mRNA accumulation analyses (Fig. 6 B). Mutant and
wild-type plants again differ with respect to the read-through transcription product initiating upstream of ycf3
or aadA. The association of these read-through transcripts
with polysomes demonstrates that they are efficient substrates for the chloroplast translation machinery. This
finding is in accordance with the results of earlier studies
showing that both monocistronic and polycistronic mRNAs
are efficiently translated in higher plant chloroplasts (2,
34). No difference in polysome loading could be detected
between mutant and wild-type plants suggesting that translation of psaA and psaB is initiated with comparable efficiencies in wild-type and We have also tested polysome association for psaC (Fig.
7 B) and, as a control, for a tetracistronic PSII transcript
(psbE/F/L/J; data not shown). These analyses also failed
to provide evidence for any defect in polysome loading in
The recent development of facile methods of transformation for higher plant chloroplasts has enabled us to address
functional aspects of plastid open reading frames by reverse genetics. In the course of this work, we have performed the first targeted inactivation of a tobacco plastid
open reading frame of unknown function by deleting the
intron-containing ycf3 from the chloroplast genome. We
have shown that homoplasmic Several lines of evidence suggest that none of the general processes in plastid gene expression (i.e., transcription, RNA processing, translation) are impaired in What then is the cause of the PSI-deficient phenotype?
And consequently, what is the function of the ycf3 gene
product? Our Northern blot and polysome association
analyses suggest that neither transcription of PSI genes,
nor transcript processing or translation initiation is impaired in The lack of evidence for a transcriptional or posttranscriptional role of the ycf3 gene product is consistent with
the idea that the control of these steps in chloroplast gene
expression is probably exclusively exerted by nuclear factors (for review see references 9, 11). We, therefore, propose that ycf3 encodes a factor involved in the assembly of
a stable PSI unit in a posttranslational fashion. This could
be the case if the Ycf3 protein is an integral part of PSI or
alternatively, if it served as an auxiliary factor for the assembly or stability of the PSI complex in the thylakoid membrane. Both possibilities imply that the absence of
virtually all PSI subunits from Isolated cyanobacterial PSI complexes consist of 11 polypeptides (PsaA, B, C, D, E, F, I, J, K, L, and M) (for
review see reference 21), which all are well characterized
at the molecular level. In view of the presence of a cyanobacterial ycf3 homologue (39), it therefore appears unlikely that the ycf3 gene product is an integral component
of the PSI complex. In this light, our results may be more
consistent with the idea that the Ycf3 protein serves as an
assembly or stability factor for PSI. However, at present
we do not know the exact suborganellar localization of the
ycf3 gene product since all our attempts to raise Ycf3-specific antibodies have failed.
Several PSI mutants have been described for cyanobacteria and Chlamydomonas reinhardtii. Insertional inactivation of psaC in C. reinhardtii was shown to result in destabilization of PSI and the concomitant loss of all PSI
subunits (38). Given our failure to detect PsaC protein in
In conclusion, our results indicate that the chloroplast
ycf3 reading frame is indeed a functional gene. Its gene
product is a heretofore unknown factor involved in the
generation of functional PSI units. Future analyses will
aim to determine the localization of the Ycf3 protein and
to define the nature of its association or interaction with
other components of photosystem I.
Fig. 1.
Experimental strategy for targeted replacement of the
ycf3 reading frame. (A) Map of the plastid DNA region containing ycf3. Genes above the line are transcribed from left to right;
genes below the line are transcribed in the opposite direction.
Restriction sites relevant for vector construction, RFLP analysis,
or generation of hybridization probes are marked. Introns are
shown as open boxes. (B) Map of the plastid DNA fragment in
the final transformation vector pSR2. A chimeric spectinomycin
resistance gene (aadA) replaces ycf3. Restriction sites eliminated
by ligation with different half-sites are shown in parentheses.
Note that the aadA gene is transcribed in the same direction as
ycf3 in the cognate sequence of the plastid genome.
[View Larger Version of this Image (14K GIF file)]
MATERIALS AND METHODS
-AACCTCCTATAGACTAGGC-3
-AGCGAAATGTAGTGCTTACG-3
-ATGTCACATTCAGTAAAGAT-3
-TCAATAAGCTAGACCCATAC-3
-CCCTTCTATGACAAATTTGA-3
-CCAGCGGATCTAAACAATCT-3
-GGTTTTTCAATGCGAGATCTA-3
-CATGACAATAACTAGAATGAA-3
ycf3 Plastid Transformation Vector
-untranslated region (nucleotide position 46,424). The BsmBI site is located close to the end of the ycf3 coding region, 17 nucleotides upstream of the termination codon. After a fill-in reaction of the
recessed ends with Klenow DNA polymerase, a chimeric aadA gene conferring resistance to aminoglycoside antibiotics (36) was inserted to replace ycf3 and to facilitate selection of chloroplast transformants. A plasmid clone carrying the aadA gene in the same orientation as previously
ycf3 yielded the final transformation vector pSR2 (see Fig. 1 B).
-untranslated region of the chimeric aadA gene) and P11 (derived from the 3
portion of the aadA coding region). Three independent transplastomic lines were subjected to
four additional rounds of regeneration on RMOP/spectinomycin to obtain
homoplasmic tissue. Homoplasmy was verified by DNA gel blot analysis
(see Fig. 2).
Fig. 2.
RFLP analysis to
verify chloroplast transformation and homoplasmy of the
ycf3 plants. Total cellular
DNA from wild-type plants and from three independently
transformed lines (Nt-pSR2-1,
Nt-pSR2-2, and Nt-pSR2-5, subsequently referred to as 2-1, 2-2, and 2-5) was digested with XhoI and hybridized to the radiolabeled SacI/XhoI fragment covering the region downstream of the
ycf3 reading frame (i. e., the psaA gene and the 5
portion of
psaB; Fig. 1). The probe detects a 5.6-kb fragment in wild-type
plants (corresponding to nucleotide positions 40,883 to 46,524;
29; Fig. 1) and a 4.9-kb fragment in the transplastomic lines. Absence of the 5.6-kb signal in the lanes representing the
ycf3
plants indicates a uniformly transformed population of plastid
DNA molecules.
[View Larger Version of this Image (18K GIF file)]
[32P]dATP-labeled probes
were generated by random priming (Boehringer Mannheim, Mannheim,
Germany) following the instructions of the manufacturer. A radiolabeled
SacI/XhoI restriction fragment (corresponding to nucleotide positions
43,807-40,883 in the tobacco chloroplast genome) (29) was used as probe
for the restriction fragment-length polymorphism (RFLP) analysis. Tobacco psaC-, psaI-, and psaJ-specific probes were synthesized by radiolabeling PCR products covering the entire coding regions of the genes (obtained by amplification with primer pair P31/P32 for psaC, P33/P34 for
psaI, and P35/P36 for psaJ). A psaA probe was prepared from an internal
NdeI fragment (corresponding to nucleotide positions 41,479-42,376). Hybridizations were carried out at 65°C in Rapid Hybridization Buffer (Amersham Intl.). A restriction fragment covering the entire coding region was used as an aadA-specific probe.
RESULTS
ycf3 Plants Exhibit a Photosynthetically
Deficient Phenotype
ycf3 lines displayed a pale-green phenotype upon regeneration on spectinomycin-containing medium under standard light conditions (3.5-4
W/m2). When transferred to boxes (for rooting on drug-
and phytohormone-free medium), the plants bleached out
completely within a few days (Fig. 3 A). The phenotype
was much less severe under low light conditions (0.4-0.5
W/m2). The plants were now light green (Fig. 3 B), and
nearly indistinguishable from wild-type plants kept under
identical conditions. However, the mutant plants grew
very slowly, and after maintenance for more than 6 wk the
lower leaves began to turn white. Only young leaves (up to
3-wk-old) from plants grown under low light conditions were used for the following molecular analyses.
Fig. 3.
Phenotype of homoplasmic ycf3 plants. (A) A mutant
plant kept under standard light conditions (3.5-4 W/m2). Massive
photooxidative damage in mutant chloroplasts results in completely white plants. (B) A mutant plant grown under low light
conditions (0.4-0.5 W/m2). Bars, 1 cm.
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ycf3 plants can be
attributed to a specific complex in the thylakoid membrane we performed immunoblot analyses using various
antibodies raised against proteins of PSII, PSI, the cytochrome bf complex, and the plastid ATP synthase complex
(Table I). Whereas PSII proteins as well as cytochrome bf
complex and ATPase subunits are readily detected in thylakoid membrane protein preparations from
ycf3 plants,
PSI proteins appear to be absent or accumulate to levels
falling below the sensitivity of our Western blots (Fig. 4).
PSI subunits are also undetectable in the soluble protein fraction excluding the possibility that the proteins are stable in the stroma but cannot be incorporated into the thylakoid membrane.
ycf3 Plants by Immunoblot Analyses
Fig. 4.
Accumulation of thylakoid proteins in ycf3 plants.
Immunoblots probed with antisera against the ATPase subunit
AtpB, the PSII proteins PsbA,
PsbD, PsbO, PsbP, and Lhcb6,
the cytochrome bf complex subunit PetA, and the PSI proteins
PsaC, PsaD, and PsaF are shown
for wild-type plants and two
or three independently transformed
ycf3 lines. For comparison, a dilution series of the
wild-type extract is shown. Chlorophyll concentrations were wild
type (higher concentration; first
lane)/wild type (lower concentration; second lane)/mutant (1:
0.2:1.) Note that PSI proteins are undetectable in mutant
plants whereas all the other protein complexes of the thylakoid
membrane appear to be not primarily affected by the absence
of the ycf3 gene product.
[View Larger Version of this Image (15K GIF file)]
ycf3 plants (data not shown). Thus it seems
that the lack of ycf3 gene product selectively compromises
PSI and does not primarily affect any of the other photosynthetic protein complexes.
Fig. 5.
Fluorescence measurements as test for PSII activity in ycf3 plants. Wild-type and mutant plants
grown under low light conditions were dark adapted, and
leaf samples were illuminated with white actinic light.
As a control, a wild-type
sample treated with the plastoquinone-reducing herbicide 3-(3,4-dichlorphenyl)-
1,1-dimethylurea (DCMU)
was included. PSII activity is
clearly detectable in
ycf3 plants. However, comparison
of the variable fluorescent
yields indicates that the mutant accumulates fewer functional PSII reaction centers
than the wild type, which
is most likely the result of photooxidative damage as
caused by the lack of functional electron acceptors
downstream of PSII. The
course of the fluorescence
curve recorded for the mutant is virtually identical with the one
of the wild-type sample treated with DCMU demonstrating that
in both cases electrons accumulate in PSII and are not transferred to downstream components of the photosynthetic electron
transport chain.
[View Larger Version of this Image (13K GIF file)]
ycf3 plants can be envisaged: (a) plastid-encoded PSI genes are not transcribed, (b) their mRNAs
are not stable, or (c) not translated. Also, the ycf3 gene
product could play a posttranscriptional role in either (d)
PSI assembly or (e) stability.
ycf3 plants.
Fig. 6.
Northern blot analysis to test transcript patterns and
mRNA accumulation in wild-type and homoplasmic transformed
ycf3 plants. Total plant RNA was hybridized to probes specific
for psaC (A), psaA (B), psaI (C), and psaJ (D). The major transcripts of ~0.5 kb for psaC (18), 5.2 kb for psaA and psaB (17),
0.6 kb for psaI, and 0.5 kb for psaJ, respectively, are marked by
arrows. No significant differences in mRNA accumulation between wild-type and mutant plants could be detected, thus excluding a pretranslational cause of the PSI-deficient phenotype.
Note a difference in the size of a minor RNA species detected by
the psaA-specific probe (asterisks; 6.9-kb transcript in mutant
plants). This RNA species represents a polycistronic transcript
initiating far upstream of psaA. The polymorphism thus reflects
the size difference of ycf3 in wild type versus the chimeric aadA
gene in mutant plastids. (The diffuse signal in the wild-type lane
(wt) is due to the presence of splicing intermediates of the intron-containing ycf3 gene, which give rise to multiple bands.) Read-through transcription, as the cause of the appearance of these
high molecular weight mRNA species, was verified by hybridizing the blot with an aadA-specific probe (B, right panel). This
probe detects the same 6.9-kb transcript as the psaA-specific
probe in
ycf3 plants and, in addition, the 1.0-kb monocistronic
aadA transcript (and a 1.4-kb aadA transcript stabilized by the
downstream 3
-UTR of the deleted ycf3 gene).
[View Larger Version of this Image (55K GIF file)]
ycf3 mutant lines demonstrating that replacement of ycf3 with aadA does not interfere with transcription of the downstream psaA and psaB genes. This is in
good agreement with the earlier finding that the tobacco
psaA/B genes are independently transcribed from their
own promoter as shown by capping analysis (17). However, wild-type and mutant lines differ in the size of a minor RNA species. This RNA species is the result of read-through transcription initiating upstream of ycf3 and
aadA, respectively (Fig. 6 B). Thus the transcript-length polymorphism is merely caused by the size difference of
the larger ycf3 in wild type versus the smaller aadA in mutant plastid genomes.
ycf3 plastids excluding a
role of the ycf3 gene product in PSI mRNA synthesis or
maturation.
ycf3 Plants
ycf3 plastids, formally two possibilities remain: (a) the ycf3 gene product plays a cotranslational
role, i.e., ycf3 encodes an essential PSI gene-specific translation factor; or (b) the ycf3 gene product is posttranslationally involved in the assembly of PSI subunits into a stable complex. To distinguish between these two possibilities
we set out to test whether or not transcripts of PSI genes
are translated in
ycf3 plastids.
ycf3
plastids by in organello translation assays. Analysis of the
polysomal association of PSI mRNAs is therefore the
method of choice to test for faithful translation initiation
on PSI transcripts in
ycf3 plants. Wild-type and mutant leaf samples were lysed under conditions maintaining the
integrity of polysomes (3). The lysates were then fractionated in sucrose gradients, and the distribution of chloroplast transcripts was analyzed by performing Northern hybridization experiments with RNA purified from gradient
fractions. As a control, EDTA was added to a gradient
containing lysate from mutant plants. EDTA treatment releases ribosomes from mRNAs. Comparison of EDTA-containing with EDTA-free gradient fractions thus allows
for the identification of monosome- versus polysome-containing fractions (Fig. 7).
Fig. 7.
Test for association
of PSI gene transcripts with
polysomes in ycf3 plants.
(A) RNAs extracted from
fractions 2-5 of analytical polysome isolation gradients
were separated on 1% formaldehyde-containing agarose
gels, transferred to nylon
membranes, and hybridized
to a psaA-specific probe
mainly detecting the dicistronic psaA/B transcripts.
Comparison of EDTA-free
with EDTA-containing gradient fractions identifies fractions 2 and 3 as mainly
monosome containing, and
fractions 4 and 5 as polysome
containing. The psaA/B transcripts in
ycf3 plastids are
as efficiently associated with
polysomes as in wild-type
plastids (Wt). Note the prominent band for the read-through transcript initiating
upstream of the aadA
marker gene in mutant plastids (compare with Fig. 6 B),
which is translated with extraordinarily high efficiency (most probably owing to the strong
[rbcL-derived] Shine-Dalgarno sequence of the chimeric aadA).
Transcript sizes are given at the right. The direction of polysome
sedimentation is marked by horizontal arrows below the blot. (B)
Analysis of polysome association for psaC transcripts. As psaA/B
mRNAs, psaC transcripts are loaded with ribosomes with comparable efficiencies in wild-type and mutant plastids. The polysome-associated monocistronic psaC transcript (horizontal arrow) is predominantly present in fractions 3 and 4 for both wild-type and mutant plastids, but nearly exclusively in fraction 2 of the EDTA-containing gradient. (C) Ribosome content of the fractions collected. RNA aliquots of the fractions were separated under nondenaturing conditions on 2% agarose gels stained with ethidium bromide. The ribosome-containing fractions show
prominent bands representing the ribosomal RNA species.
[View Larger Version of this Image (37K GIF file)]
ycf3 plants.
ycf3 plastids.
DISCUSSION
ycf3 plants display a pigment-deficient phenotype, most probably caused by the
complete absence of PSI.
ycf3
plants. First, homoplasmic mutant plants display a high
level of resistance to spectinomycin indicating that the chimeric aadA gene is highly expressed in the transgenic plastids. Second, the protein products of those plastid-encoded
photosynthesis genes that are not related to PSI can be
readily detected in mutant plastids. This finding also confirms that all of the chloroplast-encoded genes engaged in
transcription or translation provide functional gene products in
ycf3 plastids. Third, transcripts of plastid-encoded
PSI genes are faithfully synthesized, correctly processed,
accumulate to wild-type levels in
ycf3 plants and are also
efficiently loaded with ribosomes.
ycf3 plastids. Efficient loading with polysomes
is suggestive of active translation of PSI mRNAs in mutant
plastids. Identical distribution patterns of PSI transcripts across the polysome gradients also indicate that the numbers of ribosomes associated with PSI mRNAs do not significantly differ between wild-type and mutant plastids
suggesting that translation elongation proceeds with comparable efficiencies. However, these data do not completely exclude a deficiency in a late step in PSI gene-specific translation elongation (or termination) in
ycf3 plastids.
ycf3 plastids is a secondary
consequence of the destabilization of PSI caused by the
missing ycf3 gene product.
ycf3 plants, it is therefore not surprising that all the other
PSI proteins tested by immunoblot analysis were not
found either. A similar crucial role in PSI stability is attributed to the two large reaction center subunits PsaA and
PsaB. In cyanobacteria (30) as well as in Chlamydomonas (10) and higher plants (25), a defective reaction center
protein leads to a complete loss of the PSI complex and to
a rapid turnover of all of its subunits.
Received for publication 5 March 1997 and in revised form 7 July 1997.
The authors wish to thank M. Hermann for excellent technical assistance, E. Schiefermayr for oligonucleotide synthesis and A. Herzfeld and M. Messerschmid for photographic work. We are grateful to P. Maliga and Z. Svab for making available ptDNA clones and the chimeric aadA gene construct and to K. Paal and P. Zeltz for valuable discussion. We are indebted to R. Oelmüller, R.B. Klösgen, and A. Barkan for generously providing antibodies. We wish to thank K. Biehler, W. Haehnel, and G. Buchholz for help with the physiological measurements, and G.L. Igloi for critical reading of this manuscript.
PSI and PSII, photosystem I and II; RFLP, restriction fragment-length polymorphism.
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