Plant Cell Illumina
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

First published online July 3, 2008; 10.1105/tpc.107.054965

The Plant Cell 20:1786-1804 (2008)
© 2008 American Society of Plant Biologists

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
20/7/1786    most recent
tpc.107.054965v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yu, F.
Right arrow Articles by Rodermel, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yu, F.
Right arrow Articles by Rodermel, S.
Agricola
Right arrow Articles by Yu, F.
Right arrow Articles by Rodermel, S.

Mutations in SUPPRESSOR OF VARIEGATION1, a Factor Required for Normal Chloroplast Translation, Suppress var2-Mediated Leaf Variegation in Arabidopsis[W]

Fei Yua, Xiayan Liua, Muath Alsheikhb, Sungsoon Parkc and Steve Rodermela,1

a Department of Genetics, Development, and Cell Biology, Iowa State University, Ames, Iowa 50011
b Graminor Breeding AS, Bjørke Forsøksgård, Hommelstadvegen 60, N-2344 Ilseng, Norway
c Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, California 94720

1 Address correspondence to rodermel{at}iastate.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
 REFERENCES
 
The Arabidopsis thaliana yellow variegated2 (var2) mutant is variegated due to lack of a chloroplast FtsH-like metalloprotease (FtsH2/VAR2). We have generated suppressors of var2 variegation to gain insight into factors and pathways that interact with VAR2 during chloroplast biogenesis. Here, we describe two such suppressors. Suppression of variegation in the first line, TAG-FN, was caused by disruption of the nuclear gene (SUPPRESSOR OF VARIEGATION1 [SVR1]) for a chloroplast-localized homolog of pseudouridine ({Psi}) synthase, which isomerizes uridine to {Psi} in noncoding RNAs. svr1 single mutants were epistatic to var2, and they displayed a phenotypic syndrome that included defects in chloroplast rRNA processing, reduced chloroplast translation, reduced chloroplast protein accumulation, and elevated chloroplast mRNA levels. In the second line (TAG-IE), suppression of variegation was caused by a lesion in SVR2, the gene for the ClpR1 subunit of the chloroplast ClpP/R protease. Like svr1, svr2 was epistatic to var2, and clpR1 mutants had a phenotype that resembled svr1. We propose that an impairment of chloroplast translation in TAG-FN and TAG-IE decreased the demand for VAR2 activity during chloroplast biogenesis and that this resulted in the suppression of var2 variegation. Consistent with this hypothesis, var2 variegation was repressed by chemical inhibitors of chloroplast translation. In planta mutagenesis revealed that SVR1 not only played a role in uridine isomerization but that its physical presence was necessary for proper chloroplast rRNA processing. Our data indicate that defects in chloroplast rRNA processing are a common, but not universal, molecular phenotype associated with suppression of var2 variegation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 METHODS
 REFERENCES
 
Variegation mutants are ideal models to gain insight into the mechanisms of chloroplast biogenesis (reviewed in Rodermel, 2002Go; Sakamoto, 2003Go; Yu et al., 2007Go). A growing number of variegation mutants have been characterized at the molecular level, primarily in Arabidopsis thaliana, maize (Zea mays), and tomato (Solanum lycopersicum) (reviewed in Yu et al., 2007Go). Our laboratory has focused on Arabidopsis variegations, one of which is yellow variegated2 (var2) (Martínez-Zapater, 1993Go; Chen et al., 1999Go). Sectoring in var2 is due to the action of a nuclear recessive mutation. Whereas cells in the green leaf sectors and cotyledons of var2 contain morphologically normal chloroplasts, cells in the yellow and white sectors are heteroplastidic and contain vacuolated plastids with few organized lamellae in addition to some normal-appearing chloroplasts (Chen et al., 1999Go).

VAR2 codes for a chloroplast FtsH metalloprotease (designated VAR2/FtsH2) (Chen et al., 2000Go; Takechi et al., 2000Go). This protein belongs to a large class of AAA (for ATPase associated with various cellular activities) proteins that are ubiquitous among prokaryotes and eukaryotes. AAA proteins contain one or more AAA cassette domains (~200 to 250 amino acids), which act as ATP-powered molecular motors to drive processes of protein denaturation, disassembly, and translocation (Sauer et al., 2004Go). It is thought that proteins destined for destruction by FtsH are denatured by the AAA cassette, allowing degradation by the proteolytic domain to occur (Sauer et al., 2004Go).

In Arabidopsis, there are 12 FtsH genes (Sokolenko et al., 2002Go; Sakamoto et al., 2003Go; Yu et al., 2004Go). All are located in chloroplasts except FtsH3, -4, and -10, which are targeted to mitochondria (Lindahl et al., 1996Go; Chen et al., 2000Go; Sakamoto et al., 2002Go, 2003Go; Yu et al., 2004Go); FtsH11 appears to be dual-targeted to both chloroplasts and mitochondria (Urantowka et al., 2005Go). Chloroplast FtsH proteins are present in thylakoid membrane complexes that are composed (at a minimum) of two phylogenetic pairs of proteins that are at least partially functionally redundant (viz, FtsH2/8 and FtsH1/5). FtsH2 and -8 are interchangeable with one another, as are FtsH1 and -5 (Sakamoto et al., 2003Go; Yu et al., 2004Go, 2005Go; Zaltsman et al., 2005bGo). However, FtsH2 and -8 are not interchangeable with FtsH1 and -5.

The functions of FtsH have been most extensively investigated in Escherichia coli, in which both chaperone and protease activities have been identified (Suzuki et al., 1997Go). Chloroplast FtsH mediates a variety of functions that are important for chloroplast biogenesis and maintenance (Chen et al., 1999Go; Zaltsman et al., 2005aGo; Yu et al., 2007Go). The best understood of these is a role in the D1 turnover process, during which FtsH participates in the degradation of photodamaged D1 proteins of photosystem II (PSII) (Lindahl et al., 2000Go; Bailey et al., 2002Go; Sakamoto et al., 2002Go; Silva et al., 2003Go; Chen et al., 2006Go; Yoshioka et al., 2006Go).

We have proposed a threshold model to explain the mechanism of var2 variegation (Yu et al., 2004Go, 2007Go). In this model, a threshold of FtsH-containing complexes is required for normal chloroplast function and green sector formation. This model has received widespread support from morphological, biochemical, and double mutant studies (Zaltsman et al., 2005bGo; Kato et al., 2007Go). To better understand VAR2 function and the underlying mechanism of var2 leaf variegation, we have conducted genetic suppressor screens and isolated a number of mutants that modify the var2 variegation phenotype (Park and Rodermel, 2004Go). Our long-range goal is to use var2 suppressors/enhancers as a tool to gain understanding of the pathways and processes that regulate chloroplast biogenesis. Toward this goal, we plan to generate a comprehensive genetic interactions map, based on var2 suppressors, and then use this map to provide guidance in the design of experiments to test hypotheses about mechanisms of chloroplast development. The utility of this approach is illustrated by our demonstration that var2 variegation can be suppressed by mutations in ClpC2, the gene for a chloroplast Hsp100 chaperone (Park and Rodermel, 2004Go). This provided evidence of a linkage between FtsH and Clp protease function in chloroplasts, thus opening up possibilities for future research into chloroplast protease networks.

Here, we characterize two var2 suppressors generated by T-DNA tagging (designated TAG-FN and TAG-IE). We show that mutation of a chloroplast-localized pseudouridine synthase, designated SUPPRESSOR OF VARIEGATION1 (SVR1), is responsible for the suppression of variegation in the TAG-FN line. This enzyme isomerizes uridine to pseudouridine ({Psi}) in noncoding RNAs. Suppression of variegation in the second line (TAG-IE) is due to mutation of SVR2, a nuclear gene for the ClpR1 subunit of the chloroplast ClpP/R protease. This is thus another example of linkage between FtsH and Clp protease function in chloroplasts. The suppressor lines, as well as the svr1 and svr2 single mutants, condition defects in chloroplast rRNA processing, chloroplast translation, and chloroplast protein accumulation. We present a model of var2 variegation suppression whereby reduced rates of plastid protein synthesis decrease the demand for FtsH in developing chloroplasts, allowing more plastids to overcome a threshold and turn green in a var2 background. Confirmation of this model comes from the finding that variegation is suppressed by growth of var2 in the presence of chemical inhibitors of chloroplast translation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 METHODS
 REFERENCES
 
Isolation of var2 Suppressors by T-DNA Activation Tagging
We previously reported the isolation of var2 suppressors using ethyl methanesulfonate mutagenesis (Park and Rodermel, 2004Go). Here, we used T-DNA activation tagging (Weigel et al., 2000Go) as an alternative mutagenesis approach. This was accomplished by transforming plasmid pSKI015 (activation tagging vector) into var2-5, a weak var2 allele (Chen et al., 2000Go). We observed a number of lines in which the T1 and/or T2 generations had altered variegation phenotypes due to gain-of-function or loss-of-function mutations. Figures 1A and 1B show a line (designated TAG-FN) in which the T1 plant was variegated but the T2 plants were variegated or all green in an ~3:1 ratio (variegated:nonvariegated). This indicated that suppression of variegation in this line was due to a recessive (loss-of-function) mutation. We named this locus SVR1; the mutant allele in TAG-FN was designated svr1-1. TAG-FN plants grew somewhat more slowly than wild-type plants (Figure 1A), but they attained a normal stature at flowering. Mature leaves of the TAG-FN plants had normal chlorophyll concentrations, chlorophyll a/b ratios (Figures 1C and 1D), and chloroplast anatomies (see Supplemental Figure 1 online). These observations suggested that chloroplast biogenesis was not grossly altered in the TAG-FN suppressor line.


Figure 1
View larger version (40K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 1. Phenotype of TAG-FN, a var2-5 Suppressor.

(A) Representative 4-week-old wild-type, var2-5, and TAG-FN plants. The plants were maintained at 22°C under continuous light (100 µmol·m–2·s–1).

(B) Enhanced magnification of a portion of var2-5 ([A]), illustrating the extent of leaf variegation; var2-5 is a weak allele (low amount of variegation).

(C) and (D) Chlorophyll contents (on a fresh weight basis) (C) and chlorophyll a/b ratios (D) in leaves from 2-week-old wild-type, var2-5, and TAG-FN seedlings. Rosette leaves were pooled from individual plants for each determination. Each bar represents an average ± SD of three different pooled leaf samples.

 
Identification of the Suppressor Gene in TAG-FN
To identify the suppressor gene in TAG-FN, we performed cosegregation analysis to establish the linkage between the T-DNA insert(s) and the mutant phenotype. DNA gel blot analyses revealed that a 6.5-kb HindIII band cosegregated with the suppression-of-variegation phenotype (see Supplemental Figure 2 online). This band was isolated by plasmid rescue, and the rescued plasmid was sequenced. We recovered a 92-bp Arabidopsis genomic sequence flanking the T-DNA right border in the rescued plasmid that corresponded to a portion of intron 6 of At2g39140 on chromosome 2. This suggested that At2g39140 was SVR1. At2g39140 contains nine exons and eight introns (Figure 2A ) and has been annotated as the gene for a 410–amino acid protein (45.1 kD) that bears resemblance to members of the E. coli pseudouridine synthase family of enzymes.


Figure 2
View larger version (36K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 2. Identification of the Suppressor Gene in TAG-FN.

(A) The location of the T-DNA insert in At2g39140 is shown. This gene is designated SVR1, and the insertion allele is designated svr1-1. Boxes represent exons and lines denote introns. The T-DNA insert is not drawn to scale. The triangle shows the location of the conserved nine–amino acid sequence in the active site (see Figure 6B). RB, right border.

(B) Representative RNA gel blot of SVR1 mRNA accumulation in leaves from 4-week-old wild-type, var2-5, and TAG-FN seedlings (as in Figure 1A). Rosette leaves were pooled from individual plants for each determination. Equal amounts of total leaf RNA (8 µg) were electrophoresed through formaldehyde gels and transferred to nylon filters; the filters were probed with 32P-labeled SVR1 cDNA. Arrows show the sizes of the wild-type versus two smaller SVR1 mRNAs (mutant) in TAG-FN. Ethidium bromide staining of rRNAs is shown as a loading control.

(C) Full-length SVR1 transcript accumulation in the wild type and TAG-FN. Total leaf RNA was extracted and reverse-transcribed. SVR1 cDNA was detected using primers that flank the SVR1 gene. Amplification of ACTIN2 served as a control. The primers are listed in Supplemental Table 1 online.

 

Figure 6
View larger version (18K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 6. Phylogenetic Analysis of Pseudouridine Synthases.

(A) Phylogenetic tree of pseudouridine synthases from E. coli (Tru, Rlu, and Rsu gene products), Arabidopsis (At gene products), and Chlamydomonas (Maa2 gene product). Full-length protein sequences were obtained from the National Center for Biotechnology Information and The Arabidopsis Information Resource, aligned, and analyzed using MEGA 2.1 (Kumar et al., 2001Go). Genes for 17 pseudouridine synthase–like proteins were found in the Arabidopsis genome; five of these had chloroplast targeting sequences (C) and five had mitochondrial targeting sequences (M). Targeting sequences were identified using TargetP and ChloroP (Emanuelsson et al., 2007Go). All 17 are expressed at the level of mRNA. The SVR1 (At2g39140) gene product is boxed, and bootstrap values are shown at nodes. Supplemental Data Set 1 online contains the alignment used to generate this tree.

(B) An active site Asp residue is perfectly conserved among pseudouridine synthases from all sources, including 11 enzymes from E. coli, 10 from yeast, Maa2 from Chlamydomonas, SVR1 from Arabidopsis, and dyskerin from human. The Asp is embedded within a nine–amino acid sequence that is also highly conserved. All protein sequences were retrieved from GenBank, and the graph was constructed using WebLogo 3 (Crooks et al., 2004Go); the larger the letter, the more conserved the amino acid.

 
Figure 2B shows that TAG-FN (var2-5/var2-5 svr1-1/svr1-1) contained two forms of SVR1 mRNAs. The sizes of these transcripts were smaller than the mature-sized mRNAs in the wild type and var2-5 (i.e., truncated SVR1-1 transcripts were produced in TAG-FN). This conclusion was confirmed by RT-PCR analyses showing that full-length SVR1 transcripts did not accumulate in TAG-FN (Figure 2C). It is possible, therefore, that svr1-1 translation products retained function (at least in part) and that svr1-1 was a knockdown, rather than a knockout, allele. The subsequent isolation of a more severe SVR1 allele (svr1-2; described in Figure 5 below) lent support to this conclusion.


Figure 5
View larger version (24K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 5. Identification of svr1-2, a Molecular Null Allele.

(A) Salk_013085 contains a T-DNA insertion in the first exon of At2g39140 (SVR1); we designated this line svr1-2.

(B) Phenotypes of wild-type and homozygous svr1-1 and svr1-2 plants.

 
SVR1 Is the var2 Suppressor Locus: Complementation of TAG-FN
Complementation tests were performed to confirm that suppression of var2 variegation in TAG-FN was due to the disruption of SVR1 expression. Because TAG-FN was homozygous recessive at the SVR1 and VAR2 loci (svr1-1/svr1-1 var2-5/var2-5), we reasoned that overexpression of a wild-type copy of SVR1 in TAG-FN might mask the svr1 defect, thus restoring a variegation phenotype. To test this hypothesis, we transformed TAG-FN with an overexpression construct that contained a full-length SVR1 cDNA driven by the cauliflower mosaic virus (CaMV) 35S promoter. Consistent with our hypothesis, the transgenic plants were variegated (Figures 3A and 3B ). This was likely a consequence of SVR1 overexpression, inasmuch as the transformants had dramatically increased SVR1 transcript levels (Figure 3C). These data support the notion that a defect in SVR1 was responsible for the suppression of var2-5 variegation in the TAG-FN double mutants.


Figure 3
View larger version (44K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 3. Complementation of TAG-FN.

(A) TAG-FN was transformed with a construct containing SVR1 driven by the CaMV 35S promoter (TAG-FN P35S:SVR1; designated the TAG-FN complementation line). A representative T2 plant is shown. Controls are var2-5 and TAG-FN.

(B) Enhanced magnification of a portion of the TAG-FN complementation line (boxed in [A]) showing that the leaves are variegated.

(C) Representative RNA gel blot of SVR1 mRNA accumulation in var2-5, TAG-FN, and the TAG-FN complementation line (conducted as in Figure 2B).

 
Genetic Interactions between svr1-1 and Alleles of var2
To obtain svr1-1 single mutants (i.e., svr1-1/ svr1-1 VAR2/VAR2 seedlings), we backcrossed TAG-FN to wild-type Columbia and selfed the F1; the F2 were genotyped using derived cleaved amplified polymorphic sequence primers that detected mutant var2-5 sequences (Park and Rodermel, 2004Go). Figure 4A shows that svr1-1 single mutants were not variegated and that they resembled TAG-FN in morphology and growth habit. This indicated that svr1-1 was epistatic to var2-5.


Figure 4
View larger version (82K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 4. Genetic Interactions between TAG-FN and Two var2 Alleles.

(A) TAG-FN was crossed with the wild type, and svr1-1/svr1-1 single mutants were isolated in the F2; they resembled TAG-FN. The svr1-1 single mutants were crossed with var2-4: the F1 plants (var2-4/var2-4 svr1-1/svr1-1) were not variegated, indicating that svr1-1 was able to suppress the variegation phenotype of var2-4. Shown are the wild type, var2-4, the svr1-1 single mutants (i.e., svr1-1/svr1-1 seedlings), and the double mutants (var2-4/var2-4 svr1-1/svr1-1). var2-5 and TAG-FN are also shown for comparison.

(B) Enhanced magnification of the boxed portion of (A) showing that var2-4 is a more severe allele than var2-5.

 
The var2-5 allele contains a missense mutation in the AAA cassette domain of the protein (Chen et al., 2000Go). It is a leaky allele and accumulates VAR2 mRNA and protein; its variegation phenotype is less severe than that of var2-4, a putative null allele (Chen et al., 2000Go) (Figures 4A and 4B). var2-4 lacks detectable VAR2 mRNA and protein due to a splice site mutation. To examine the allele specificity of svr1-1–mediated variegation suppression, we crossed svr1-1 with var2-4. Figure 4A shows that the double mutants (svr1-1/svr1-1 var2-4/var2-4) were not variegated and that they resembled TAG-FN. This indicated that the mechanism of svr1-1 suppression was not allele-specific and, in addition, that suppression of variegation did not require the presence of the VAR2 protein.

Identification of svr1-2, a Molecular Null Allele
Because we could not rule out the possibility that svr1 was a leaky allele, we sought to identify a true knockout allele of this gene in the Salk collection of T-DNA insertion mutants (http://signal.salk.edu/cgi-bin/tdnaexpress; Alonso et al., 2003Go). Several lines were identified, one of which (Salk_013085) had an insertion in the first exon of the gene (Figure 5A ). Homozygous plants of this line (designated svr1-2) were yellow-green and significantly reduced in size compared with wild-type and svr1-1 plants (Figure 5B). The leaves of svr1-2 also remained pale green throughout their development.

Supplemental Figure 3B online shows that svr1-2 was able to suppress the variegation phenotypes of var2-5 (leaky allele) and var2-4 (null allele); both double mutants resembled svr1-2 single mutants. This confirmed (1) that loss-of-function mutations in SVR1 were epistatic to var2 and (2) that interactions between SVR1 and VAR2 proteins were not required for the suppression of var2 variegation phenotypes.

Taken together, the observations in Figure 5 and Supplemental Figure 3B online indicated that svr1-2 was a more severe allele than svr1-1. Consistent with this idea, SVR1 transcripts could not be detected in this line by RNA gel blot analysis (data not shown). This indicated that svr1-2 was a molecular null allele.

SVR1 Encodes a Pseudouridine Synthase
As mentioned earlier, At2g39140 has been annotated as a member of the Arabidopsis pseudouridine synthase gene family. {Psi} is the most abundant modified nucleotide in noncoding RNA species, including rRNAs, tRNAs, small nuclear RNAs, and small nucleolar RNAs; it is not normally found in mRNAs (Charette and Gray, 2000Go; Ofengand, 2002Go). The isomerization of uridine to {Psi} is catalyzed by pseudouridine synthase (Charette and Gray, 2000Go).

Phylogenetic analyses were undertaken as a first step toward determining whether SVR1 was a bona fide pseudouridine synthase. Pseudouridine synthases are best characterized in E. coli, in which there are 11 family members. These enzymes mediate uridine-to-{Psi} isomerization in tRNAs (designated Tru genes) and rRNAs (designated Rlu genes, for ribosomal 50S rRNAs, and Rsu genes, for ribosomal 30S rRNAs) (Koonin, 1996Go; Ofengand, 2002Go; Kaya and Ofengand, 2003Go). E. coli pseudouridine synthases diverge widely in amino acid sequence, yet they fold into similar structures (Mueller, 2002Go). They also share a conserved nine–amino acid motif at the active site that includes a universally conserved Asp residue. This Asp is perfectly conserved not only among E. coli pseudouridine synthases but also among all other pseudouridine synthases identified to date, ranging from bacteria to yeast to humans (Ofengand, 2002Go). The frequency of amino acids within this motif in pseudouridine synthases from diverse sources is illustrated in Figure 6B .

To determine the complement of pseudouridine synthases in Arabidopsis, we searched the databases for sequences with similarity to each of the 11 E. coli proteins. Twenty-two homologous sequences were found. Of these, 17 contained the active site motif with the perfectly conserved Asp; these included SVR1. The phylogenetic relatedness of the E. coli, Arabidopsis, and Chlamydomonas pseudouridine synthases is illustrated in Figure 6A. The tree is complex but notable for the relative lack of phylogenetic pairs that have arisen as a consequence of duplications that occurred during the evolution of the Arabidopsis genome, as seen in many Arabidopsis gene families, including FtsH (Yu et al., 2007Go). On the basis of our phylogenetic analyses, we concluded that SVR1 bore all of the hallmarks of a true pseudouridine synthase.

SVR1 Is Localized in the Plastid
SVR1 was predicted to be plastid-localized based on the presence of a putative transit peptide of 35 amino acids. To confirm this localization, we examined the intracellular location of a transiently expressed P35S:SVR1:GFP (for green fluorescent protein) fusion construct in protoplasts isolated from wild-type Arabidopsis leaves; the control was a P35S:GFP fusion construct. Fusion protein expression was monitored by confocal microscopy. Figures 7A to 7C show control protoplasts that were transformed with GFP alone: green fluorescence was present in the cytoplasm (Figure 7A) but was not associated with the chloroplast (as shown by chlorophyll autofluorescence; Figure 7B); consistent with this apparent lack of congruity, the two fluorescence images did not merge (Figure 7C). By contrast, green fluorescence from the SVR1:GFP fusion protein (Figure 7D) matched the pattern of chlorophyll autofluorescence (Figure 7E); this was confirmed by the merging of the two images (Figure 7F). These data indicated that SVR1 was a chloroplast protein.


Figure 7
View larger version (44K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 7. Localization and Expression of SVR1.

(A) to (F) Leaf protoplasts were isolated from wild-type Arabidopsis leaves and transiently transformed with a control GFP vector ([A] to [C]) or a SVR1:GFP vector ([D] to [F]). Green fluorescence from GFP ([A] and [D]) and chlorophyll autofluorescence ([B] and [E]) were monitored by confocal microscopy. (C) and (F) show merged images of (A) and (B) and (D) and (E), respectively. Each panel shows a single protoplast.

(G) Expression of SVR1 mRNAs was examined in the indicated tissues from wild-type plants by RNA gel blot analysis (performed as in Figure 2B). The filter was probed with 32P-labeled SVR1 cDNAs. The ethidium bromide–stained gel is shown as a loading control.

 
To determine whether SVR1 is expressed in plastid types other than chloroplasts, we conducted RNA gel blot analyses using RNAs from various tissue types (Figure 7G). As anticipated, SVR1 was highly expressed in chloroplast-containing tissues; we suspect that floral expression was from the sepals. SVR1 mRNAs also accumulated in amyloplast-containing root tissues but not in etioplast-containing tissues of dark-grown seedlings. We concluded that SVR1 was important for chloroplast development and maintenance but that it likely functioned in other plastid types as well.

Defects in SVR1 Mediate Alterations in Chloroplast rRNA Processing
rRNAs are the most abundant RNA species in plant cells, and plastid rRNAs (23S, 16S, 5S, and 4.5S) and cytosolic rRNAs (25S, 18S, 5.8S, and 5S) can be easily observed on ethidium bromide–stained, denaturing agarose gels (Leaver and Ingle, 1971Go; Kössel et al., 1985Go). Casual observation of RNA gels such as those in Figures 2 and 3 revealed that chloroplast rRNA metabolism was likely affected in the TAG-FN suppressor line, inasmuch as the abundant 1.2- and 1.0-kb 23S rRNA species were reduced in amount compared with the wild type and var2-5 (Figure 2).

To examine the patterns of chloroplast rRNA accumulation in TAG-FN in greater detail, we conducted RNA gel blot analyses using gene-specific probes. The genes for chloroplast rRNAs (23S, 16S, 5S, and 4.5S) are located in the large inverted repeat region of land plant chloroplast genomes, where they form part of the rrn operon (Figure 8A ). This operon, which also includes two tRNA genes, is transcribed as a large polycistronic RNA that is processed by endoribonucleases and exoribonucleases to yield the mature rRNA and tRNA species (Bollenbach et al., 2005Go).


Figure 8
View larger version (37K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 8. Chloroplast rRNA Processing Is Altered in TAG-FN.

(A) The structure of the chloroplast rRNA (rrn) operon in Arabidopsis. In addition to four rRNA genes, the operon contains genes for tRNA-I (Ile) and tRNA-A (Ala); each tRNA gene has a large intron. tRNA-V (Val) is immediately upstream from the 5' transcription start site of the operon, marked by the arrow. The probes used in the RNA gel blot analyses in (B) are shown as thick black lines under the individual rRNA genes.

(B) Accumulation of chloroplast rRNAs. RNA gel blot experiments were conducted as in Figure 2B using equal amounts of total cell RNAs (2 µg) from leaves of 4-week-old wild-type, var2-5, TAG-FN, and the TAG-FN P35S:SVR1 complementation line. The blots were probed with gene-specific sequences for plastid rRNAs (see [A]) and for the cytosolic 18S rRNA; the primers for these probes are listed in Supplemental Table 1 online. Ethidium bromide staining (rRNA control) of each gel is shown in Supplemental Figure 7 online. Sizes and precursor identifications are in agreement with previous reports (Bollenbach et al., 2005Go).

 
Figure 8B shows that there was a dramatic shift in the stoichiometry of rRNAs complementary to the 23S probe in TAG-FN compared with the wild type, var2-5, and the TAG-FN complementation line. This shift was due to a marked enhancement in levels of the three largest 23S rRNAs (the 3.2-kb precursor and the 2.9- and 2.4-kb mature rRNA species) and to a sharp decline in levels of the four smaller 23S rRNA species (the 1.7-kb processing intermediate and the mature 0.5-, 1.0-, and 1.2-kb rRNA species). Maturation of the 4.5S, 5S, and 16S rRNAs was also perturbed in TAG-FN: levels of the mature rRNAs were decreased, while those of the precursor rRNAs were increased. Cytosolic 18S rRNA levels were normal in all plants, suggesting that the accumulation of cytosolic rRNAs was not affected in the mutants.

Considered together, the data in Figure 8B indicated that TAG-FN was impaired in chloroplast rRNA processing and that this defect was rescued in the TAG-FN complementation line. This supported the idea that defects in chloroplast rRNA processing can be mediated by svr1-1. This was confirmed by examining profiles of chloroplast rRNA accumulation in the svr1-1 and svr1-2 single mutants. Supplemental Figure 3A online shows that these profiles were qualitatively the same as those in TAG-FN (Figure 8B) but that there were quantitative differences in rRNA accumulation between svr1-1 and svr1-2. In particular, svr1-2 had enhanced accumulation of the three largest 23S rRNAs and of the 16S and 4.5S rRNA precursors as well as compensating decreases in the levels of the four smaller 23S rRNAs and of the mature 16S and 4.5S rRNAs. These observations were consistent with the notion that svr1-2 was a more severe allele than svr1-1.

Nuclear and Plastid Gene Expression in svr1 Mutants
One question that arose was whether the alterations in plastid rRNA processing were indicative of a more global defect in RNA metabolism in svr1 plants. Were patterns of mRNA accumulation affected? To address this question, we performed RNA gel blot analyses using representative nuclear and plastid genes as probes. These included rbcL (encoding the ribulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco] large subunit; LS), psbA (encoding the D1 protein of PSII), psaA (encoding the PsaA protein of PSI), atpA (encoding the {alpha}-subunit protein of the proton ATP synthase), and petB (encoding cytochrome f of the cytochrome b6f complex). Two nuclear genes for plastid proteins served as controls: RbcS (encoding the Rubisco small subunit) and Lhcb2 (encoding the major light-harvesting chlorophyll a/b binding protein of PSII).

Figure 9A shows that the transcript profiles of the two nuclear genes were quantitatively and qualitatively similar in the wild type, var2-5, TAG-FN, and the TAG-FN complementation line. The patterns of mRNA accumulation were also qualitatively similar for each of the plastid genes. This indicated that mRNA processing per se was not likely altered in TAG-FN, since genes that were transcribed as polycistronic mRNAs, then processed to their mature forms (such as atpA and petB), had qualitatively similar mRNA accumulation profiles. It was notable, however, that plastid mRNA levels were increased in TAG-FN by about twofold, as estimated by comparing the amount of rbcL mRNA in TAG-FN to a titration series of rbcL mRNA accumulation in wild-type plants (Figure 9B). These increases were mediated by svr1-1, inasmuch as they were reversed in the TAG-FN complementation line. Considered together, the data in Figure 9 suggested that SVR1 played a global role in plastid mRNA accumulation, perhaps by affecting rates of transcription and/or turnover.


Figure 9
View larger version (39K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 9. Patterns of Nuclear and Chloroplast mRNA Accumulation.

(A) Expression of representative nuclear and plastid genes in the wild type, var2-5, TAG-FN, and the TAG-FN complementation line. Total cell RNA was isolated from leaves of 3-week-old seedlings grown at 22°C under continuous light (100 µmol·m–2·s–1). RNA gel blot analyses with the indicated probes were conducted as in Figure 2B. Ethidium bromide staining of each RNA gel is shown in Supplemental Figure 7 online.

(B) Total cell RNA from the wild type and TAG-FN was loaded on an agarose gel in a dilution series, and RNA gel blot analysis was conducted using an rbcL probe.

 
We were next interested in determining whether a lack of SVR1 affected chloroplast protein accumulation. For these experiments, we conducted immunoblot studies (Figure 10 ) on leaf proteins from the wild type, var2-5, TAG-FN, svr1-1, and svr1-2 using antibodies against representative photosynthetic proteins: D1, PsbP, and Lhcb2 (all from PSII); PsaF (from PSI); the Rieske Fe-S protein (from the cytochrome b6f complex); ATP{alpha} (the {alpha}-subunit of the proton ATPase); and LS. We also probed the blots with antibodies against VAR2 and FtsH1.


Figure 10
View larger version (72K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 10. Patterns of Protein Accumulation.

Immunoblot analyses were conducted on total cell proteins from leaves of the wild type, var2-5, TAG-FN, svr1-1, and svr1-2; the plants were grown as in Figure 9. Following electrophoresis, the proteins were transferred to a nitrocellulose membrane and probed with polyclonal antibodies against the proteins indicated. The wild-type samples were loaded in a dilution series, and other lanes were standardized to equal fresh weights of leaf tissue.

 
Consistent with the nearly normal chlorophyll levels and chlorophyll a/b ratios in TAG-FN and var2-5 (Figures 1C and 1D), the data in Figure 10 showed that chloroplast protein levels were very similar in var2-5, TAG-FN, and svr1-1. Exceptions were VAR2 and FtsH1, which were dramatically reduced in amount in var2-5 and TAG-FN. This was as expected for var2-5: FtsH1, FtsH2 (VAR2), FtsH5, and FtsH8 interact to form oligomeric complexes in the thylakoid membrane, and when one of these proteins is produced in excess, it is degraded to match the abundance of the others (Yu et al., 2004Go, 2005Go). The fact that VAR2 and FtsH1 were coordinately reduced in amount in TAG-FN indicated that suppression of var2 variegation in this line was not due to an enhancement of VAR2 and/or of FtsH1 protein abundance.

In contrast with svr1-1, some of the plastid proteins in Figure 10 were markedly reduced in amount. For instance, we consistently observed reductions of ~50% in D1, PsaF, the Rieske Fe-S protein, ATP{alpha}, and LS. This suggested that SVR1 was capable of influencing chloroplast protein accumulation.

Inhibition of Chloroplast Translation Suppresses var2 Variegation
The data in Figures 9 and 10 were consistent with the idea that defects in SVR1 resulted in higher than normal chloroplast mRNA levels but decreased abundance of at least some chloroplast proteins. This suggested that translation was impaired in the svr1 mutants. This might not be unexpected, since chloroplast rRNA processing defects of the sort documented for svr1 (Figure 8; see Supplemental Figure 3 online) have previously been correlated with defects in plastid ribosome assembly and translation (Bollenbach et al., 2005Go; Koussevitzky et al., 2006Go). To test this hypothesis, we pulse-labeled wild-type and svr1-2 plants with [35S]Met for 20 min. In this system, the LS is the most abundant labeled protein in the soluble fraction and the D1 protein of PSII is the most abundant labeled protein in the membrane fraction (Rodermel et al., 1988Go; Pesaresi et al., 2001Go). Figures 11A and 11B show that the amounts of labeled LS and D1 that accumulate during the pulse were markedly reduced in svr1-2. This was consistent with the notion that chloroplast translation was reduced in svr1-2.


Figure 11
View larger version (63K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 11. Chloroplast Translation and Suppression of var2 Variegation.

(A) and (B) Pulse-labeling of chloroplast proteins. Young, expanding leaves from wild-type and svr1-2 seedlings were pulse-labeled with [35S]Met for 20 min. Total soluble proteins (A) or membrane proteins (B) were extracted, and equal cpm of trichloroacetic acid–precipitable proteins were subjected to SDS-PAGE analysis. Labeled bands were detected by autoradiography. Rubisco LS was the most prominent labeled membrane protein, and the D1 protein of PSII was the most prominent labeled membrane protein.

(C) Translation inhibitor experiments. var2-4 seedlings were grown in the presence or absence of chloramphenicol (10 mM CAP) or spectinomycin (1 mg/L Spec). var2 cotyledons are green, while the true leaves are variegated (Chen et al., 1999Go).

 
To test whether translation defects were able to suppress var2 variegation, we treated var2-4 (a null allele) with chemical inhibitors of chloroplast translation, including chloramphenicol, which prevents the binding of amino acyl–tRNA to the A site on the 50S subunit of 70S ribosomes (Pestka, 1971Go), and spectinomycin, which prevents translocation of the peptidyl-tRNA from the A site to the P site on the 30S subunit of 70S ribosomes (Vázquez, 1979Go). We found that high concentrations of these inhibitors were lethal but that sublethal concentrations were able to suppress the variegation phenotype of var2-4 (Figure 11C).

Mutagenesis of the Perfectly Conserved Asp in the Active Site of SVR1
As mentioned earlier, pseudouridine synthases contain a short stretch of nine amino acids in their active sites that includes a perfectly conserved Asp residue (Koonin, 1996Go; Ofengand, 2002Go) (Figure 6B). Mutations of this residue abolish the ability of pseudouridine synthase to isomerize uridine to {Psi} in E. coli (Ofengand, 2002Go). This Asp corresponds to amino acid 274 of the SVR1 gene product.

We wanted to take advantage of our molecular null allele (svr1-2) to ask whether in vitro mutagenized copies of SVR1 that contained substitutions of this residue could complement the svr1-2 defect, thus normalizing the mutant phenotype. Figure 12A shows results from a representative experiment in which Asp-274 was mutagenized in vitro to Asn (D274N), a substitution of a charged residue with a similarly sized, uncharged residue. The mutant form of SVR1 was transformed into svr1-2 under the control of the CaMV 35S promoter; the transformants were designated P35S:SVR1(D274N). Transformation of svr1-2 with a wild-type SVR1 cDNA served as a control (transformants were designated P35S:SVR1). Figure 12A shows that both sets of transgenic plants resembled the wild type (i.e., both constructs were able to rescue svr1-2). This correlated with high levels of mRNA expression from both transgenes (Figure 12B). Figure 12B further shows that the 23S rRNA processing defects in svr1-2 were reversed in the overexpressors. This is consistent with our earlier data showing that SVR1 is necessary for normal chloroplast rRNA processing.


Figure 12
View larger version (34K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 12. Site-Directed Mutagenesis of the Conserved Asp-247 of SVR1.

(A) Representative 3-week-old wild-type, svr1-2, svr1-2 transformed with a P35S-driven wild-type copy of SVR1 (P35S:SVR1), and svr1-2 transformed with a P35S-driven mutant SVR1, designated P35S:SVR1(D274N), plants. In the mutant, Asp-274 was altered to Asn.

(B) RNA gel blot analysis of RNA from leaves of the plants in (A). The experiments were conducted as in Figure 2B; the blot was probed with labeled SVR1 sequences. The arrows label the two bands corresponding to 23S RNA, and the ethidium bromide–stained gel is shown as a loading control.

(C) Representative 3-week-old wild-type, var2-5, TAG-FN, and TAG-FN transformed with the P35S-driven mutant SVR1 construct, designated P35S: SVR1(D274N), plants.

(D) RNA gel blot analysis of RNA from leaves of the plants in (C). The experiment was conducted as in Figure 2B; the blot was probed with the 23S rRNA sequences described in Figure 8A. The ethidium bromide–stained gel of rRNAs is shown in Supplemental Figure 7 online.

 
Figure 3 shows that overexpression of wild-type SVR1 was able to restore a variegated phenotype to the TAG-FN suppression line. To test whether the active site Asp-274 was necessary for this restoration, we transformed TAG-FN with the D274N construct. Figure 12C shows that the transformants were variegated. This suggested that D274N, like wild-type SVR1, was able to complement the svr1-1 defect in TAG-FN. Also like the TAG-FN lines in which wild-type SVR1 was overexpressed (Figure 8B), the 23S rRNA-processing defect in TAG-FN was normalized in the D274N overexpression lines (Figure 12D). This indicated that Asp-274, which is required for the isomerization of uridine to {Psi}, was not required for plastid rRNA processing. However, plastid rRNA processing appeared to require the presence of the SVR1 protein per se (Figure 12B).

Another var2 Suppressor, svr2, Is Impaired in Chloroplast rRNA Processing
During the course of our var2 suppressor screens, we identified a T-DNA–tagged line (designated TAG-IE) with a chloroplast rRNA processing defect similar to that of svr1 (Figure 13E ). TAG-IE plants were smaller than normal and had a pronounced virescent phenotype, with newly emerged leaves having a bright yellow coloration (Figure 13A). We found that TAG-IE suppressed var2 in a recessive manner and that the TAG-IE suppressor phenotype cosegregated with a complex T-DNA structure (see Supplemental Figure 4 online). DNA gel blot analyses revealed that this structure was likely composed of multiple rearranged T-DNAs. We named the suppressor locus SVR2 and the mutant allele in TAG-IE svr2-1. Homozygous svr2-1 plants had the same virescent phenotype as TAG-IE, indicating that svr2-1 was epistatic to var2-5 in TAG-IE.


Figure 13
View larger version (34K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 13. Another var2 Suppressor, SVR2, Affects Chloroplast rRNA Processing.

(A) Representative 4-week-old wild-type, var2-5, and TAG-IE (var2-5 svr2-1) plants.

(B) The gene structure of ClpR1/SVR2. The three sets of PCR primers used to identify the site of the T-DNA insertion are shown. The insertion is complex and resides between primers F2 and R2.

(C) Determination of the T-DNA insertion site in ClpR1/SVR2. Genomic DNA was extracted from TAG-IE, and PCR was performed using three sets of overlapping primers to the Arabidopsis ClpR1 gene (Koussevitzky et al., 2006Go), as indicated in (B). The primers F2 and R2 were not able to amplify DNA, indicating that the insertion resided between them. The arrow designates an internal PCR control (see Methods).

(D) The expression of ClpR1/SVR2 mRNA in the wild type and TAG-IE. Total cell RNA was extracted from the wild type and TAG-IE and subjected to RT-PCR analysis using the F1 and R3 primers; these primers should amplify a full-length ClpR1/SVR2 cDNA.

(E) RNA gel blot analysis of 23S rRNA accumulation in the wild type, var2-5, and TAG-IE. The blotting was performed as in Figure 2B using total leaf RNA from 3-week-old seedlings; the blot was probed with 23S RNA sequences.

 
Due to the complex nature of the T-DNA insertion in svr2-1, we failed to isolate the gene via plasmid rescue and thus turned to map-based cloning procedures. Bulked segregant analysis of a mapping population obtained by crossing TAG-IE with Landsberg erecta showed that svr2-1 was genetically linked to molecular markers ciw1 and nga80 on chromosome 1. Using a candidate gene approach, we noted that this region of chromosome 1 contained genes for a number of putative and identified plastid proteins, including At1g49970, the gene for the ClpR1 subunit of the chloroplast Clp protease complex. It has been suggested that Clp protease core complexes (325 to 350 kD), which are composed of ClpP/R/S subunits (Peltier et al., 2001Go), are involved in general protein turnover in plastids (Adam and Clarke, 2002Go; Adam et al., 2006Go). We focused our attention on ClpR1 because previously described clpR1-1 and clpR1-2 mutants have a virescent phenotype similar to that of svr2-1 (Koussevitzky et al., 2006Go).

To test whether svr2-1 defined the gene for ClpR1, we performed allelism tests between svr2-1 and clpR1-2, a T-DNA insertion allele (Salk_088407) (Koussevitzky et al., 2006Go). We found that clpR1-2 and svr2-1 failed to complement one another, suggesting that they were allelic. To confirm this hypothesis, we examined whether the ClpR1 gene in TAG-IE contained a T-DNA structure. This was accomplished by PCR analysis of genomic DNA from TAG-IE using three pairs of primers that spanned the Arabidopsis ClpR1 gene (Figure 13B). Figure 13C shows that the F2 and R2 primer pair failed to amplify a genomic DNA fragment, consistent with the idea that an insertion resided between these two primers. We also failed to detect a ClpR1 transcript in TAG-IE via RT-PCR using ClpR1 primers that would have amplified the entire gene (Figure 13D). This indicated that the clpR1 allele in TAG-IE was likely a molecular null. Considered together, the data in Figure 13 supported the idea that SVR2 encoded ClpR1.

Molecular characterization of clpR1-1 (a null allele) has been performed in the Chory laboratory (Koussevitzky et al., 2006Go). Their studies showed that this mutant has many similarities to svr1. First, it accumulates higher molecular weight forms of chloroplast 23S and 4.5S rRNAs, suggesting that it is impaired in chloroplast rRNA processing. Second, it has normal levels of mRNA from several representative plastid genes but reduced protein accumulation. To explain these findings, Koussevitzky et al. (2006)Go suggested that chloroplast rRNA maturation and translation are linked and that chloroplast translation is inhibited in clpR1-1. Again, this is similar to our conclusions regarding svr1-2.

Are Plastid rRNA Defects Common in var2 Suppressors?
The question arose whether defects in plastid rRNA processing were universally associated with the suppression of var2 variegation. To address this question, we examined chloroplast 23S rRNA profiles in various suppressors of var2 we had generated (see Methods); the suppressors described below were all recessive. Supplemental Figure 5 online shows that three suppressors had profiles that resemble the wild type (and var2-5): TAG-11, ems2505, and ems2544. By contrast, other suppressor lines had 23S rRNA processing defects similar to those in TAG-FN, svr1-2, and TAG-IE. These included clpC1, a mutant that lacks the ClpC1 chaperone, which could suppress var2 variegation at early stages of seedling development (see Supplemental Figure 6 online). We concluded that not all var2 suppressors had defects in chloroplast rRNA processing.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
METHODS
 REFERENCES
 
Chloroplast rRNA Processing and Translation Are Perturbed in svr1 and svr2
The molecular players that mediate plastid rRNA processing are poorly understood, but they include two 3' to 5' exoribonucleases. One is a polynucleotide phosphorylase (PNPase) that is involved in 23S rRNA processing as well as the metabolism of tRNAs and mRNAs (Walter et al., 2002Go; Sauret-Güeto et al., 2006Go), and the other is a homolog of E. coli RNase R (RNR1) that is involved in the maturation of 23S, 16S, and 5S rRNAs (Kishine et al., 2004Go; Bollenbach et al., 2005Go). Other factors that affect chloroplast rRNA maturation are defined by a handful of mutants that accumulate rRNA intermediates. These include the following: (1) maize high chlorophyll fluorescence7 (Barkan, 1993Go), maize rnc1 (Watkins et al., 2007Go), and Arabidopsis white cotyledon (Yamamoto et al., 2000Go), all of which accumulate primarily 16S rRNA precursors; (2) Chlamydomonas ac20, which is defective in 23S rRNA maturation (Holloway and Herrin, 1998Go); (3) Arabidopsis dal (for dag-like, from the differentiation and greening mutant of snapdragon [Antirrhinum majus]) (Chatterjee et al., 1996Go), which accumulates 16S and 23S precursor rRNAs (Babiychuk et al., 1997Go; Bisanz et al., 2003Go); and (4) tomato dcl (for defective chloroplasts and leaves), in which 4.5S rRNA processing is defective (Bellaoui et al., 2003Go); 4.5S rRNA processing is also impaired in Arabidopsis mutants with downregulated DCL gene expression (Bellaoui and Gruissem, 2004Go).

These mutants define genes whose products affect chloroplast rRNA processing. Several of these genes have been cloned, including DCL and DAL, which code for novel chloroplast proteins (Bellaoui et al., 2003Go; Bisanz et al., 2003Go), and RNC1, which codes for a maize group II splicing factor that mediates RNA binding but does not have endonuclease activity (Watkins et al., 2007Go). Mutants deficient in RNC1 (rnc1) are illustrative of the principle that mutants with defects in chloroplast rRNA processing need not define genes whose products are directly involved in this process; rather, processing defects are frequently found in mutants with primary lesions in ribosome assembly and/or function (Keus et al., 1984Go; Barkan, 1993Go; Leal-Klevezas et al., 2000Go; Bellaoui et al., 2003Go; Williams and Barkan, 2003Go; Bollenbach et al., 2005Go; Schmitz-Linneweber et al., 2006Go). An early demonstration of this principle (Shen and Bremer, 1977Go) was the observation that rRNA maturation is defective in E. coli cells treated with chloramphenicol, an inhibitor of translation on 70S ribosomes. In a complementary manner, PNPase and RNR1 mutants, which have primary defects in chloroplast rRNA processing, have pleiotropic effects that include reduced rates of chloroplast translation and protein accumulation (Walter et al., 2002Go; Bollenbach et al., 2005Go; Sauret-Güeto et al., 2006Go).

One of the distinguishing molecular phenotypes of svr1 is its impairment in the processing of all four plastid rRNA species (23S, 16S, 5S, and 4.5S). These defects were accompanied by above-normal levels of chloroplast mRNAs but by decreased rates of chloroplast protein synthesis and by reduced accumulation of chloroplast proteins. We conclude that svr1 mediates defects in chloroplast rRNA processing either directly or indirectly. We propose that a similar explanation applies to svr2 (Koussevitzky et al., 2006Go).

It might be noted that the defects in chloroplast rRNA processing in svr1 were accompanied by chloroplast mRNA levels that exceeded those found in the wild type; rbcL mRNA levels, for example, were approximately doubled (Figure 9). Close inspection of the RNA gel blot data described by Koussevitzky et al. (2006)Go reveals that the levels of some chloroplast mRNAs are also elevated in clpR1-1. Although we do not understand the reason for these increases, our data are consistent with Barkan's (1993)Go early observation that the accumulation of chloroplast mRNA species is frequently altered in mutants with chloroplast ribosome defects.

Possible Role of SVR1 in Chloroplast RNA Metabolism
In this report, we found that suppression of var2 variegation in TAG-FN is caused by the disruption of SVR1, the gene for a plastid pseudouridine synthase. The isomerization of uridine-to-{Psi} occurs posttranscriptionally, and although the precise function of {Psi} in noncoding RNAs is unclear, it has been proposed to play a structural role and might also be involved in peptidyl transfer during translation (Lane et al., 1992Go; Charette and Gray, 2000Go; Ofengand, 2002Go).

One of the central findings of the research reported here is that mutation of the conserved Asp in the active site of SVR1 does not affect chloroplast rRNA processing but that correct rRNA processing requires SVR1 protein accumulation (Figure 12). Our working hypothesis is that Asp is required to isomerize uridine to {Psi} but that SVR1 per se is necessary for rRNA processing. For instance, SVR1 could be involved in RNA binding as a component of a ribonucleoprotein complex that directly or indirectly affects rRNA metabolism. In support of this idea, the chloroplast Maa2 (for maturation of psaA) pseudouridine synthase from Chlamydomonas is necessary for trans splicing of group II introns in chloroplast mRNAs as well as for the isomerization of uridine (Perron et al., 1999Go). However, site-directed mutagenesis of several well-conserved amino acids of Maa2 (including the active site Asp) revealed that isomerization activity is not required for trans splicing, but splicing requires the physical presence of the protein (Perron et al., 1999Go). Similar results were reported for E. coli TruB and RluD pseudouridine synthase mutants (Gutgsell et al., 2000Go, 2001Go, 2005Go) as well as for yeast rRNA pseudouridine synthase Cbf5p (Lafontaine et al., 1998Go). By analogy to Maa2, it is thus possible that the reductions in translation that we observed in svr1 plants might be due to a defect in splicing of a chloroplast mRNA for a protein required for chloroplast ribosome assembly/function. It might be noted that in addition to pseudouridine synthases, other examples of enzymes that modify noncoding RNAs and whose functions extend beyond this modification activity include E. coli methyltransferases RlmAI (Liu et al., 2004Go), a tRNA m5U54-methyltransferase (Persson et al., 1992Go), and rRNA methylases from yeast (the DIM1 gene product) and Arabidopsis chloroplasts (the PALEFACE1 gene product) (Lafontaine et al., 1995Go; Tokuhisa et al., 1998Go).

Based on phylogenetic considerations, SVR1 bears all of the hallmarks of a bona fide pseudouridine synthase. However, we have not directly demonstrated that the enzyme has isomerization activity. Even if it is ultimately demonstrated that SVR1 does not have pseudouridine synthase activity, our data clearly suggest that the presence of the protein is required for normal rRNA processing. Such a situation would be consistent with the idea that enzymes involved in plastid RNA metabolism are frequently derived from antecedents with different roles in RNA metabolism. A well-known example of this phenomenon is RNC1, which is a component of a chloroplast ribonucleoprotein complex that promotes the splicing of group II introns (Watkins et al., 2007Go). RNC1 contains two ribonuclease III domains, but amino acids that are essential for ribonuclease III activity are missing in the protein. Mutant and biochemical analyses have revealed that the role of RNC1 in splicing involves RNA binding but not endonucleolytic activity. Another example of this general phenomenon is CRS2, a maize group II intron splicing factor that lacks its ancestral peptidyl-tRNA hydrolase activity (Ostheimer et al., 2005Go).

Model of Suppression of var2 Variegation
Suppression of var2 variegation by svr1 and svr2 is likely to be indirect, inasmuch as our genetic analyses showed that svr1-2 and svr2-1 are able to suppress var2-4 (a null allele). This suggests that the physical presence of the VAR2 protein is not necessary for suppression; this does not rule out an involvement of SVR1 or SVR2 with other components of the FtsH complex. Taken together, our data suggest that suppression of variegation in both svr1 and svr2 is caused by a phenotypic syndrome that includes defects in chloroplast rRNA processing, reduced chloroplast translation, reduced chloroplast protein accumulation, and elevated chloroplast mRNA levels. We propose that there is a fundamental interaction between chloroplast translation and var2-mediated leaf variegation. This was confirmed by treatment of var2 with chloroplast translation inhibitors such as chloramphenicol and spectinomycin, both of which are able to suppress variegation (Figure 11).

One hypothesis of variegation suppression proceeds from the premise that a reduction in the rate of chloroplast translation prolongs the process of chloroplast biogenesis, allowing more time to accumulate a threshold of factors that are able to compensate for a lack of var2 (i.e., before the decision is made to turn white or green). (Intermediate plastid types are not seen in var2 tissues [Chen et al., 1999Go]). For example, these factors could enhance photoprotection or other var2-mediated plastid developmental processes. We propose that this prolongation would be especially important during early leaf development, when plastid divisions are occurring and proplastids are being converted into chloroplasts (Yu et al., 2004Go).

A second hypothesis is that the crucial element in variegation suppression is chloroplast translation per se and that reduced translation affects the expression of nuclear genes and/or plastid genes. It is well established that reductions in plastid translation are sensed by the nucleus via a poorly defined retrograde signaling pathway (Oelmüller et al., 1986Go; Adamska, 1995Go; Gray et al., 1995Go, Yoshida et al., 1998Go; Sullivan and Gray 1999Go, 2002Go; Sugimoto et al., 2004Go). In this pathway, reductions in chloroplast translation elicit the transmission of a signal from the plastid to the nucleus that results in altered transcription of nuclear genes, primarily those for chloroplast proteins. Therefore, it is possible that suppression of var2 variegation is caused by an altered chloroplast proteome that allows the developing plastid to compensate for a lack of VAR2.

In a third scenario, variegation suppression is caused by reduced translation of a chloroplast DNA-encoded factor. For example, this factor could be a substrate of VAR2 that normally acts to promote early chloroplast biogenesis but that later inhibits this process if it is not degraded. The inhibitor would be present in var2 (causing white sector formation), but its production would be reduced to such an extent in svr1 and svr2 plants that normal chloroplast biogenesis could proceed even in the presence of small amounts of the inhibitor. This hypothesis, like the others, would be consistent with the idea of a threshold (e.g., some plastids would have reached a threshold of the plastid factor, while others would not have).

Previous work from our laboratory demonstrated that mutations in the nuclear gene for ClpC2, a chloroplast Hsp100 chaperone, are potent suppressors of var2 variegation (Park and Rodermel, 2004Go). It has been suggested that ClpC2 functions both as a regulatory subunit of the Clp protease complex and independently as a molecular chaperone (Adam et al., 2006Go; Sakamoto, 2006Go). Our finding that mutations in another subunit of the Clp protease (ClpR1) are able to suppress var2-mediated variegation establishes a second linkage between FtsH and Clp function in chloroplasts. Our suppressor studies to date thus open up possibilities for further research into chloroplast protease networks.

In this study, we found that defective chloroplast rRNA processing is a common, but not universal, molecular phenotype of var2 suppressors and that this phenotype is accompanied by alterations in chloroplast translation and protein accumulation. We conclude that this class of suppressor should provide valuable insight into the factors that mediate plastid rRNA maturation, ribosome assembly, and function (Manuell et al., 2004Go). In support of this idea, Sakamoto's group (Miura et al., 2007Go) recently reported that var2 variegation is suppressed by defects in chloroplast elongation factor G (SCO1 locus) and chloroplast translation initiation factor 2 (FUG1 locus).


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
REFERENCES
 
Plant Material
All Arabidopsis thaliana plants used in this study are in the Columbia ecotype background. They were grown at 22°C under continuous light of ~100 µmol·m–2·s–1. The mutant var2-4 and var2-5 alleles have been described (Chen et al., 2000Go). Suppressor lines included TAG-FN (this study), TAG-IE (this study), TAG-11 (X. Liu, F. Yu, and S. Rodermel, unpublished data), all obtained by activation tagging (below); and ems2505 (S. Park and S. Rodermel, unpublished data) and ems2544 (Park and Rodermel, 2004Go), both obtained by ethyl methanesulfonate mutagenesis. svr1-2 was identified from a Salk line (Salk_013085); a T-DNA insertion allele of svr2 was also obtained from a Salk line (Salk_088407) (Koussevitzky et al., 2006Go). The clpC1 mutant was described by Sjögren et al. (2004)Go. In some experiments, var2-4 seeds were grown on Murashige and Skoog plates in the presence or absence of inhibitors of chloroplast translation (10 mM chloramphenicol or 1 mg/L spectinomycin) under the conditions described above.

Activation Tagging
The var2-5 allele was used for the activation tagging experiments. An Agrobacterium tumefaciens strain containing the activation tagging vector pSKI015 was kindly provided by Joanne Chory at the Salk Institute. Bolting Arabidopsis plants were transformed by the Agrobacterium-mediated floral dip method (Clough and Bent, 1998Go). T1 plants were germinated on soil, and transgenic plants were selected by spraying with a 1:2000 dilution of Finale (AgrEvo), which contains 5.78% (w/v) ammonium glufosinate. The T1 plants were self-fertilized, and analyses were performed on T2 and subsequent generation plants.

Map-Based Cloning
The suppressor gene in TAG-IE was mapped by bulked segregation analysis (Lukowitz et al., 2000Go) using a pool of 112 F2 seedlings from a cross between TAG-IE and Landsberg erecta. The gene was fine-mapped using sets of codominant simple sequence length polymorphism (SSLP) markers (Bell and Ecker, 1994Go). The suppressor gene resides between SSLP markers ciw1 and nga280, described by Lukowitz et al. (2000)Go. Three pairs of PCR primers were used for confirmation of the site of T-DNA insertion in the suppressor gene (F1 and R1, F2 and R2, and F3 and R3); these are listed in Supplemental Table 1 online. PCR primers MRO11F and MRO11R were used as an internal PCR control (see Supplemental Table 1 online).

Phylogenetic Analysis
Full-length protein sequences for 17 pseudouridine synthases from Arabidopsis, 11 pseudouridine synthases from Escherichia coli, and the Maa2 pseudouridine synthase from Chlamydomonas reinhardtii were obtained from the National Center for Biotechnology Information GenBank. The sequences were aligned with ClustalX (see alignment in Supplemental Data Set 1 online), and a phylogenetic tree was constructed using default settings of MEGA 2.1 (Kumar et al., 2001Go). Bootstrap analysis was performed using 1000 trials.

Plasmid Construction and Transformation
To complement the TAG-FN mutant, a full-length At2g39140 (SVR1) cDNA was amplified by Plantium pfx DNA polymerase (Invitrogen) using primers FNCF and FNCR (see Supplemental Table 1 online). The PCR product was digested with SacI and cloned into the SacI site of pBluescript KS+. The resulting construct was sequenced to confirm the SVR1 sequence. The SacI fragment was then transferred into a modified pBI121 plasmid (Yu et al., 2004Go), and the resulting construct was transformed into Agrobacterium by electroporation. Arabidopsis transformation was performed as described above (Clough and Bent, 1998Go).

To generate a GFP-tagged SVR1 fusion protein (SVR1:GFP) for transient expression in protoplasts, At2g39140 (SVR1) cDNA was amplified using primers FNCBF and FNGFPR (see Supplemental Table 1 online) and cloned into vector pTF486 at the BamHI and NcoI sites, creating a C-terminal GFP fusion. Transcription is driven by the CaMV 35S promoter (P35S) in this vector. Protoplasts from wild-type Arabidopsis leaves were isolated and transiently transformed with the P35S:SVR1:GFP construct or, alternatively, with a GFP vector control (P35S:GFP) according to Yoo et al. (2007)Go. Fluorescence from GFP and autofluorescence from chlorophyll were monitored by confocal microscopy.

DNA and RNA Gel Blot Analyses
Genomic DNA was isolated and DNA gel blot analyses were performed as described by Wetzel et al. (1994)Go. Total cell RNA isolation from Arabidopsis leaf tissues using the Trizol RNA reagent (Invitrogen) and RNA gel blot analyses were also performed as described by Wetzel et al. (1994)Go. Primers used to generate the probes used in this work are listed in Supplemental Table 1 online. Most probes were labeled by random priming, but the 4.5S and 5S probes were labeled by the end-labeling method (Sambrook et al., 1989Go). RT-PCR was performed as described (Yu et al., 2004Go) using the primers listed in Supplemental Table 1 online.

Plasmid Rescue
For plasmid rescue, genomic DNA was extracted from 100 mg of Arabidopsis tissue and resuspended in 30 µL of deionized water (Wetzel et al., 1994Go). A 10-µL aliquot of this suspension was digested with various restriction enzymes for 8 h in 30-µL reactions, and the digests were extracted with phenol-chloroform followed by chloroform. DNA was precipitated with ethanol and resuspended in 20 µL of deionized water. This suspension was split into two 10-µL fractions and ligated overnight using T4 DNA ligase (Invitrogen) at either 14°C or room temperature. The two ligation reactions were combined and extracted with phenol-chloroform, followed by chloroform, and the DNA was precipitated with ethanol and resuspended in 10 µL of deionized water. One microliter of the final suspension was used for transformation using recombination-deficient E. coli SURE competent cells (Stratagene). Rescued plasmids were selected on Luria-Bertani plates containing 100 mg/L ampicillin.

Protein Manipulations
Arabidopsis leaves were weighed and frozen in liquid N2. Frozen leaves were then homogenized in 2x SDS buffer and incubated at 65°C for 2 h. After centrifugation at 14,000 rpm for 10 min, the supernatants were loaded onto 12% SDS polyacrylamide gels based on equal fresh weights (Yu et al., 2004Go). SDS-PAGE was performed based on Laemmli (1970)Go as described by Yu et al. (2004Go, 2005Go). Polyclonal antibodies used in this study were directed against VAR2 (Chen et al., 2000Go), FtsH1 (Yu et al., 2005Go), D1 (Yu et al., 2005Go), Rieske Fe-S protein (Yu et al., 2005Go), and the ATPase {alpha}-subunit (Yu et al., 2005Go). Antibodies against PsbP and PsaF were from Agrosera and were kindly provided by Poul Erik Jensen at the University of Copenhagen. The SuperSignal West Pico chemiluminescence kit (Pierce) was used for signal detection on the immunoblots.

Labeling of proteins in vivo was performed as described previously (Rodermel et al., 1988Go; Pesaresi et al., 2001Go). In brief, [35S]Met (50 µCi) was applied to the surface of expanding Arabidopsis leaves, and the leaves were vacuum-infiltrated for 10 s. After a 20-min pulse of the label, total proteins were isolated (as described above) and equal cpm were loaded onto 12.5% SDS polyacrylamide gels. The gels were dried using a Bio-Rad Geldryer and exposed to autoradiograph film.

Chlorophyll Analysis
The first pair of rosette leaves of 2-week-old seedlings was harvested and weighed. Leaves were ground in liquid N2, and chlorophyll was extracted with 95% ethanol. Total chlorophyll contents and chlorophyll a/b ratios were calculated as described (Lichtenthaler, 1987Go).

Site-Directed Mutagenesis
The full-length At2g39140 (SVR1) cDNA was amplified using primers FNCBF and FNCBR and cloned into pBluescript KS+ at the BamHI site. Asp-274 was then mutated in vitro using the QuikChange site-directed mutagenesis kit (Stratagene). The primers used for mutagenesis were DNF and DNR (see Supplemental Table 1 online). The wild-type and mutated forms of the SVR1 cDNA were cloned into the BamHI site of pB003 (provided by Dr. David Oliver, Iowa State University); transcription was driven by the CaMV 35S promoter in this vector. The constructs were introduced into Arabidopsis by the methods described above.

Electron Microscopy
For transmission electron microscopy, leaf samples were obtained from 7-d-old seedlings grown as described above. The samples were fixed, stained, and examined as described by Horner and Wagner (1980)Go.

Accession Numbers
Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: SVR1/At2g39140, NP_181447; ClpR1/SVR2/At1g49970, NP_564560; At1g09800, NP_172451; At1g20370, NP_564112; At1g34150, NP_564438; At1g56345, NP_176031; At1g76050, NP_177732; At1g76120, NP_565126; At1g78910, NP_178012; At2g30320, NP_180591; At3g04820, NP_187133; At3g06950, NP_187351; At3g19440, NP_188575; At3g57150, NP_191274; At4g21770, NP_193908; At5g14460, NP_196950; At5g35400, NP_198390; At5g51140, NP_199927; Maa2, CAB62387; RsuA, AAC75244; RluB, AAC74351; RluE, AAC74219; RluF, AAC76992; RluA, AAC73169; RluC, AAC74170; RluD, AAC75643; TruC, AAC75833; TruB, AAC76200; TruA, AAC75378; TruD, AAC75787.

Supplemental Data
The following materials are available in the online version of this article.

Supplemental Figure 1. The Ultrastructure of Chloroplasts in the Wild Type, var2-5, and TAG-FN.
Supplemental Figure 2. Linkage Analysis of TAG-FN Double Mutants.
Supplemental Figure 3. Characterization of svr1-2.
Supplemental Figure 4. Linkage Analysis of TAG-IE Double Mutants.
Supplemental Figure 5. Chloroplast 23S rRNA Accumulation in var2 Suppressors.
Supplemental Figure 6. The Suppression of var2-5 by clpC1.
Supplemental Figure 7. Ethidium Bromide Staining of RNA Gels.
Supplemental Table 1. Primers Used in This Study.
Supplemental Data Set 1. Alignment Used to Generate the Phylogenetic Tree in Figure 6.


   Acknowledgments
 
This work was supported by funding to S.R. from the U.S. Department of Energy, Energy Biosciences Panel (Grant DE-FG02-94ER20147).


   Footnotes
 
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Steve Rodermel (rodermel{at}iastate.edu).

[W] Online version contains Web-only data. Back

www.plantcell.org/cgi/doi/10.1105/tpc.107.054965

Received August 9, 2007; Revision received May 13, 2008. accepted May 28, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 METHODS
 REFERENCES
 
Adam, Z., and Clarke, A.K. (2002). Cutting edge of chloroplast proteolysis. Trends Plant Sci. 7: 451–456.[CrossRef][Web of Science][Medline]

Adam, Z., Rudella, A., and van Wijk, K. (2006). Recent advances in the study of Clp, FtsH and other proteases located in chloroplasts. Curr. Opin. Plant Biol. 9: 234–240.[CrossRef][Web of Science][Medline]

Adamska, I. (1995). Regulation of early light-inducible protein gene expression by blue and red light in etiolated seedlings involves nuclear and plastid factors. Plant Physiol. 107: 1167–1175.[Abstract]

Alonso, J.M., et al. (2003). Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653–657.[Abstract/Free Full Text]

Babiychuk, E., Fuangthong, M., van Montagu, M., Inze, D., and Kushnir, S. (1997). Efficient gene tagging in Arabidopsis thaliana using a gene trap approach. Proc. Natl. Acad. Sci. USA 94: 12722–12727.[Abstract/Free Full Text]

Bailey, S., Thompson, E., Nixon, P.J., Horton, P., Mullineaux, C.W., Robinson, C., and Mann, N.H. (2002). A critical role for the Var2 FtsH homologue of Arabidopsis thaliana in the photosystem II repair cycle in vivo. J. Biol. Chem. 277: 2006–2011.[Abstract/Free Full Text]

Barkan, A. (1993). Nuclear mutants of maize with defects in chloroplast polysome assembly have altered chloroplast RNA metabolism. Plant Cell 5: 389–402.[Abstract]

Bell, C.J., and Ecker, J.R. (1994). Assignment of 30 microsatellite loci to the linkage map of Arabidopsis. Genomics 19: 137–144.[CrossRef][Web of Science][Medline]

Bellaoui, M., and Gruissem, W. (2004). Altered expression of the Arabidopsis ortholog of DCL affects normal plant development. Planta 219: 819–826.[Web of Science][Medline]

Bellaoui, M., Keddie, J.S., and Gruissem, W. (2003). DCL is a plant-specific protein required for plastid ribosomal RNA processing and embryo development. Plant Mol. Biol. 53: 531–543.[CrossRef][Web of Science][Medline]

Bisanz, C., Bégot, L., Carol, P., Perez, P., Bligny, M., Pesey, H., Gallois, J.-L., Lerbs-Mache, S., and Mache, R. (2003). The Arabidopsis nuclear DAL gene encodes a chloroplast protein which is required for the maturation of the plastid ribosomal RNAs and is essential for chloroplast differentiation. Plant Mol. Biol. 51: 651–663.[CrossRef][Web of Science][Medline]

Bollenbach, T.J., Lange, H., Gutierrez, R., Erhardt, M., Stern, D.B., and Gagliardi, D. (2005). RNR1, a 3'-5' exoribonuclease belonging to the RNR superfamily, catalyzes 3' maturation of chloroplast ribosomal RNAs in Arabidopsis thaliana. Nucleic Acids Res. 33: 2751–2763.[Abstract/Free Full Text]

Charette, M., and Gray, M.W. (2000). Pseudouridine in RNA: What, where, how and why. IUBMB Life 49: 341–351.[CrossRef][Web of Science][Medline]

Chatterjee, M., Sparvoli, S., Edmunds, C., Garosi, P., Findlay, K., and Martin, C. (1996). DAG, a gene required for chloroplast differentiation and palisade development in Antirrhinum majus. EMBO J. 15: 4194–4207.[Web of Science][Medline]

Chen, J., Burke, J.J., Velten, J., and Xin, Z. (2006). FtsH11 protease plays a critical role in Arabidopsis thermotolerance. Plant J. 48: 73–84.[CrossRef][Web of Science][Medline]

Chen, M., Choi, Y., Voytas, D.F., and Rodermel, S.R. (2000). Mutations in the Arabidopsis VAR2 locus cause leaf variegation due to the loss of a chloroplast FtsH protease. Plant J. 22: 303–313.[CrossRef][Web of Science][Medline]

Chen, M., Jensen, M., and Rodermel, S.R. (1999). The yellow variegated mutant of Arabidopsis is plastid autonomous and delayed in chloroplast biogenesis. J. Hered. 90: 207–214.[Abstract/Free Full Text]

Clough, S.J., and Bent, A.F. (1998). Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16: 735–743.[CrossRef][Web of Science][Medline]

Crooks, G.E., Hon, G., Chandonia, J.M., and Brenner, S.E. (2004). WebLogo: A sequence logo generator. Genome Res. 14: 1188–1190.[Abstract/Free Full Text]

Emanuelsson, O., Brunak, S., von Heijne, G., and Nielsen, H. (2007). Locating proteins in the cell using TargetP, SignalP and related tools. Nat. Protoc. 2: 953–971.[CrossRef][Medline]

Gray, J.C., Sornarajah, R., Zabron, A.A., Duckett, C.M., and Khan, M.S. (1995). Chloroplast control of nuclear gene expression. In Photosynthesis: From Light to Biosphere, Vol. 3, P. Mathis, ed (Dordrecht, The Netherlands: Kluwer Academic Publishers), pp. 543–550.

Gutgsell, N., Englund, N., Niu, L., Kaya, Y., Lane, B.G., and Ofengand, J. (2000). Deletion of the Escherichia coli pseudouridine synthase gene truB blocks formation of pseudouridine 55 in tRNA in vivo, does not affect exponential growth, but confers a strong selective disadvantage in competition with wild-type cells. RNA 6: 1870–1881.[Abstract]

Gutgsell, N.S., Del Campo, M., Raychaudhuri, S., and Ofengand, J. (2001). A second function for pseudouridine synthases: A point mutant of RluD unable to form pseudoruidines 1911, 1915, and 1917 in Escherichia coli 23S ribosomal RNA restores normal growth to an RluD-minus strain. RNA 7: 990–998.[Abstract]

Gutgsell, N.S., Deutscher, M.P., and Ofengand, J. (2005). The pseudouridine synthase RluD is required for normal ribosome assembly and function in Escherichia coli. RNA 11: 1141–1152.[Abstract/Free Full Text]

Holloway, S.P., and Herrin, D.L. (1998). Processing of a composite large subunit rRNA: Studies with Chlamydomonas mutants deficient in maturation of the 23S-like rRNA. Plant Cell 10: 1193–1206.[Abstract/Free Full Text]

Horner, H.T., and Wagner, B.L. (1980). The association of druse crystals with the developing stomium of Capsicum annum (Solanaceae) anthers. Am. J. Bot. 67: 1347–1360.[CrossRef][Web of Science]

Kato, Y., Miura, E., Matsushima, R., and Sakamoto, W. (2007). White leaf sectors in yellow variegated2 are formed by viable cells with undifferentiated plastids. Plant Physiol. 144: 952–960.[Abstract/Free Full Text]

Kaya, Y., and Ofengand, J. (2003). A novel unanticipated type of pseudouridine synthase with homologs in bacteria, archaea, and eukarya. RNA 9: 711–721.[Abstract/Free Full Text]

Keus, R.J.A., Dekker, A.F., Kreuk, K.C.J., and Groot, G.S.P. (1984). Transcription of ribosomal DNA in chloroplasts of Spirodela oligorhiza. Curr. Genet. 9: 91–98.[CrossRef][Web of Science]

Kishine, M., Takabayashi, A., Munekage, Y., Shikanai, T., Endo, T., and Sato, F. (2004). Ribosomal RNA processing and an RNase R family member in chloroplasts of Arabidopsis. Plant Mol. Biol. 55: 595–606.[CrossRef][Web of Science][Medline]

Koonin, E.V. (1996). Pseudouridine synthases: Four families of enzymes containing a putative uridine-binding motif also conserved in dUTPases and dCTP deaminases. Nucleic Acids Res. 24: 2411–2415.[Abstract/Free Full Text]

Kössel, H., Natt, E., Strittmatter, G., Fritzsche, E., Gozdzicka-Jozefiak, A., and Przybyl, D. (1985). Structure and expression of rRNA operons from plastids of higher plants. In Molecular Form and Function of the Plant Genome, L Van Vloten-Doting, GSP Groot, and TC Hall, eds (New York: Plenum Press), pp. 183–198.

Koussevitzky, S., Stanne, T.M., Peto, C.A., Giap, T., Sjögren, L.L.E., Zhao, Y., Clarke, A.K., and Chory, J. (2006). An Arabidopsis thaliana virescent mutant reveals a role for ClpR1 in plastid development. Plant Mol. Biol. 63: 85–96.[CrossRef][Web of Science][Medline]

Kumar, S., Tamura, K., Jakobsen, I.B., and Nei, M. (2001). MEGA2: Molecular Evolutionary Genetics Analysis Software. (Tempe, AZ: Arizona State University).

Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277: 680–685.

Lafontaine, D., Vandenhaute, J., and Tollervey, D. (1995). The 18S rRNA dimethylase dim1p is required for pre-ribosomal RNA processing in yeast. Genes Dev. 9: 2470–2481.[Abstract/Free Full Text]

Lafontaine, D.L.J., Bousquet-Antonelli, C., Henry, Y., Caizergues-Ferrer, M., and Tollervey, D. (1998). The box H+ACA snoRNAs carry Cbf5p, the putative rRNA pseudouridine synthase. Genes Dev. 12: 527–537.[Abstract/Free Full Text]

Lane, B.G., Ofengand, J., and Gray, M.W. (1992). Pseudouridine in the large-subunit (23 S-like) ribosomal RNA. The site of peptidyl transfer in the ribosome? FEBS Lett. 302: 1–4.[CrossRef][Web of Science][Medline]

Leal-Klevezas, D.S., Martinez-Soriano, J.P., and Nazar, R.N. (2000). Transcription and processing map of the 4.5–5S rRNA intergenic regions (ITS3) from rapeseed (Brassica napus) chloroplasts. Plant Cell Rep. 19: 667–673.[CrossRef][Web of Science]

Leaver, C.J., and Ingle, J. (1971). The molecular integrity of chloroplast ribosomal ribonuleic acid. Biochem. J. 123: 235–243.[Web of Science][Medline]

Lichtenthaler, H.K. (1987). Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol. 148: 350–382.[CrossRef][Web of Science]

Lindahl, M., Spetea, C., Hundal, T., Oppenheim, A.B., Adam, Z., and Andersson, B. (2000). The thylakoid FtsH protease plays a role in the light-induced turnover of the photosystem II D1 protein. Plant Cell 12: 419–431.[Abstract/Free Full Text]

Lindahl, M., Tabak, S., Cseke, L., Pichersky, E., Andersson, B., and Adam, Z. (1996). Identification, characterization, and molecular cloning of a homologue of the bacterial FtsH protease in chloroplasts of higher plants. J. Biol. Chem. 271: 29329–29334.[Abstract/Free Full Text]

Liu, M., Novotny, G.W., and Douthwaite, S. (2004). Methylation of 23S rRNA nucleotide G745 is a secondary function of the RlmAI methyltransferase. RNA 10: 1713–1720.[Abstract/Free Full Text]

Lukowitz, W., Gillmor, C.S., and Scheible, W.-R. (2000). Positional cloning in Arabidopsis. Why it feels good to have a genome initiative working for you. Plant Physiol. 123: 795–805.[Abstract/Free Full Text]

Manuell, A., Beligni, M.V., Yamaguchi, K., and Mayfield, S.P. (2004). Regulation of chloroplast translation: Interactions of RNA elements, RNA-binding proteins and the plastid ribosome. Biochem. Soc. Trans. 32: 601–605.[CrossRef][Web of Science][Medline]

Martínez-Zapater, J.M. (1993). Genetic analysis of variegated mutants in Arabidopsis. J. Hered. 84: 138–140.[Abstract/Free Full Text]

Miura, E., Kato, Y., Matsushima, R., Albrecht, V., Laalami, S., and Sakamoto, W. (2007). The balance between protein synthesis and degradation in chloroplasts determines leaf variegation in Arabidopsis yellow variegated mutants. Plant Cell 19: 1313–1328.[Abstract/Free Full Text]

Mueller, E.G. (2002). Chips off the old block. Nat. Struct. Biol. 9: 320–322.[CrossRef][Web of Science][Medline]

Oelmüller, R., Levitan, I., Bergfeld, R., Rajasekhar, V.K., and Mohr, H. (1986). Expression of nuclear genes as affected by treatments acting on plastids. Planta 168: 482–492.[CrossRef][Web of Science]

Ofengand, J. (2002). Ribosomal RNA pseudouridines and pseudouridine synthases. FEBS Lett. 514: 17–25.[CrossRef][Web of Science][Medline]

Ostheimer, G.J., Hadjivassiliou, H., Kloer, D.P., Barkan, A., and Matthews, B.W. (2005). Structural analysis of the group II intron splicing factor CRS2 yields insights into its protein and RNA interaction surfaces. J. Mol. Biol. 345: 51–68.[CrossRef][Web of Science][Medline]

Park, S., and Rodermel, S.R. (2004). Mutations in ClpC2/Hsp100 suppress the requirement for FtsH in thylakoid membrane biogenesis. Proc. Natl. Acad. Sci. USA 101: 12765–12770.[Abstract/Free Full Text]

Peltier, J.-B., Ytterberg, J., Liberles, D.A., Roepstorff, P., and van Wijk, K.J. (2001). Identification of a 350-kDA ClpP protease complex with 10 different Clp isoforms in chloroplasts of Arabidopsis thaliana. J. Biol. Chem. 276: 16318–16327.[Abstract/Free Full Text]

Perron, K., Goldschmidt-Clermont, M., and Rochaix, J.-D. (1999). A factor related to pseudouridine synthases is required for chloroplast group II intron trans-splicing in Chlamydomonas reinhardtii. EMBO J. 18: 6481–6490.[CrossRef][Web of Science][Medline]

Persson, B.C., Gustafsson, C., Berg, D.E., and Björk, G.R. (1992). The gene for a tRNA modifying enzyme, m5U54-methyltransferase, is essential for viability in Escherichia coli. Proc. Natl. Acad. Sci. USA 89: 3995–3998.[Abstract/Free Full Text]

Pesaresi, P., Varotto, C., Meurer, J., Jahns, P., Salamini, F., and Leister, D. (2001). Knock-out of the plastid ribosomal protein L11 in Arabidopsis: Effects on mRNA translation and photosynthesis. Plant J. 27: 179–189.[CrossRef][Web of Science][Medline]

Pestka, S. (1971). Inhibitors of ribosome functions. Annu. Rev. Microbiol. 25: 487–562.[CrossRef][Web of Science][Medline]

Rodermel, S. (2002). Arabidopsis variegation mutants. In The Arabidopsis Book, C.R. Somerville and E.M. Meyerowitz, eds (Rockville, MD: American Society of Plant Biologists), doi/10.1199/tab.0079, http://www.aspb.org/publications/arabidopsis/.

Rodermel, S.R., Abbott, M.S., and Bogorad, L. (1988). Nuclear-organelle interactions: nuclear antisense gene inhibits ribulose bisphosphate carboxylase enzyme levels in transformed tobacco plants. Cell 55: 673–681.[CrossRef][Web of Science][Medline]

Sakamoto, W. (2003). Leaf-variegated mutations and their responsible genes in Arabidopsis thaliana. Genes Genet. Syst. 78: 1–9.[CrossRef][Web of Science][Medline]

Sakamoto, W. (2006). Protein degradation machineries in plastids. Annu. Rev. Plant Biol. 57: 599–621.[CrossRef][Medline]

Sakamoto, W., Tamura, T., Hanba-Tomita, Y., Murata, M., and Sodmergen. (2002). The VAR1 locus of Arabidopsis encodes a chloroplastic FtsH and is responsible for leaf variegation in the mutant alleles. Genes Cells 7: 769–780.[Abstract]

Sakamoto, W., Zaltsman, A., Adam, Z., and Takahashi, Y. (2003). Coordinated regulation and complex formation of yellow variegated 1 and yellow variegated 2, chloroplastic FtsH metalloproteases involved in the repair cycle of photosystem II in Arabidopsis thylakoid membranes. Plant Cell 15: 2843–2855.[Abstract/Free Full Text]

Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press).

Sauer, R.T., et al. (2004). Sculpting the proteome with AAA+ proteases and disassembly machines. Cell 119: 9–18.[CrossRef][Web of Science][Medline]

Sauret-Güeto, S., Botella-Pavía, P., Flores-Pérez, U., Martínez-García, J.F., San Román, C., León, P., Boronat, A., and Rodríguez-Concepción, M. (2006). Plastid cues posttranscriptionally regulate the accumulation of key enzymes of the methylerythritol phosphate pathway in Arabidopsis. Plant Physiol. 141: 75–84.[Abstract/Free Full Text]

Schmitz-Linneweber, C., Williams-Carrier, R.E., Williams-Voelker, P.M., Kroeger, T.S., Vichas, A., and Barkan, A. (2006). A pentatricopeptide repeat protein facilitates the trans-splicing of the maize chloroplast rps12 pre-mRNA. Plant Cell 18: 2650–2663.[Abstract/Free Full Text]

Shen, V., and Bremer, H. (1977). Chloramphenicol-induced changes in the synthesis of ribosomal, transfer, and messenger ribonucleic acids in Escherichia coli B/r. J. Bacteriol. 130: 1098–1108.[Abstract/Free Full Text]

Silva, P., Thompson, E., Bailey, S., Kruse, O., Mullineaux, C.W., Robinson, C., Mann, N.H., and Nixon, P.J. (2003). FtsH is involved in the early stages of repair of photosystem II in Synechocystis sp. PCC 6803. Plant Cell 15: 2152–2164.[Abstract/Free Full Text]

Sjögren, L.L.E., MacDonald, T.M., Sutinen, S., and Clarke, A.K. (2004). Inactivation of the clpC1 gene encoding a chloroplast Hsp100 molecular chaperone causes growth retardation, leaf chlorosis, lower photosynthetic activity, and a specific reduction in photosystem content. Plant Physiol. 136: 4114–4126.[Abstract/Free Full Text]

Sokolenko, A., Pojidaeva, E., Zinchenko, V., Panichkin, V., Glaser, V.M., Herrmann, R.G., and Shestakov, S.V. (2002). The gene complement for proteolysis in the cyanobacterium Synechocystis sp. PCC 6803 and Arabidopsis thaliana chloroplasts. Curr. Genet. 41: 291–310.[CrossRef][Web of Science][Medline]

Sugimoto, H., Kusumi, K., Tozawa, Y., Yazaki, J., Kishimoto, N., Kikuchi, S., and Iba, K. (2004). The virescent-2 mutation inhibits translation of plastid transcripts for the plastid genetic system at an early age of chloroplast differentiation. Plant Cell Physiol. 45: 985–996.[Abstract/Free Full Text]

Sullivan, J., and Gray, J.C. (1999). Plastid translation is required for the expression of nuclear photosynthesis genes in the dark and in roots of the pea lip1 mutant. Plant Cell 11: 901–910.[Abstract/Free Full Text]

Sullivan, J., and Gray, J.C. (2002). Multiple plastid signals regulate the expression of the pea plastocyanin gene in pea and transgenic tobacco plants. Plant J. 32: 763–774.[CrossRef][Web of Science][Medline]

Suzuki, C.K., Rep, M., van Dijl, J.M., Suda, K., Grivell, L.A., and Schatz, G. (1997). ATP-dependent proteases that also chaperone protein biogenesis. Trends Biochem. Sci. 22: 118–123.[CrossRef][Web of Science][Medline]

Takechi, K., Sodmergen, Murata, M., Motoyoshi, F., and Sakamoto, W. (2000). The YELLOW VARIEGATED (VAR2) locus encodes a homologue of FtsH, an ATP-dependent protease in Arabidopsis. Plant Cell Physiol. 41: 1334–1346.[Abstract/Free Full Text]

Tokuhisa, J.G., Vijayan, P., Feldmann, K.A., and Browse, J.A. (1998). Chloroplast development at low temperatures requires a homolog of DIM1, a yeast gene encoding the 18S rRNA dimethylase. Plant Cell 10: 699–711.[Abstract/Free Full Text]

Urantowka, A., Knorpp, C., Olczak, T., Kolodziejczak, M., and Janska, H. (2005). Plant mitochondria contain at least two i-AAA-like complexes. Plant Mol. Biol. 59: 239–252.[CrossRef][Web of Science][Medline]

Vázquez, D. (1979). Inhibitors of protein biosynthesis. Mol. Biol. Biochem. Biophys. 30: 178–179.

Walter, M., Kilian, J., and Kudla, J. (2002). PNPase activity determines the efficiency of mRNA 3'-end processing, the degradation of tRNA and the extent of polyadenylation in chloroplasts. EMBO J. 21: 6905–6914.[CrossRef][Web of Science][Medline]

Watkins, K.P., Kroeger, T.S., Cooke, A.M., Williams-Carrier, R.E., Friso, G., Belcher, S.E., van Wijk, K.J., and Barkan, A. (2007). A ribonuclease III domain protein functions in group II intron splicing in maize chloroplasts. Plant Cell 19: 2606–2623.[Abstract/Free Full Text]

Weigel, D., et al. (2000). Activation tagging in Arabidopsis. Plant Physiol. 122: 1003–1013.[Abstract/Free Full Text]

Wetzel, C.M., Jiang, C.Z., Meehan, L.J., Voytas, D.F., and Rodermel, S.R. (1994). Nuclear-organelle interactions: the immutans variegation mutant of Arabidopsis is plastid autonomous and impaired in carotenoid biosynthesis. Plant J. 6: 161–171.[CrossRef][Web of Science][Medline]

Williams, P.M., and Barkan, A. (2003). A chloroplast-localized PPR protein required for plastid ribosome accumulation. Plant J. 36: 675–686.[CrossRef][Web of Science][Medline]

Yamamoto, Y.Y., Puente, P., and Deng, X.-W. (2000). An Arabidopsis cotyledon-specific albino locus: A possible role in 16S rRNA maturation. Plant Cell Physiol. 41: 68–76.[Abstract/Free Full Text]

Yoo, S.D., Cho, Y.H., and Sheen, J. (2007). Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis. Nat. Protocols 2: 1565–1572.[CrossRef]

Yoshida, R., Sato, T., Kanno, A., and Kameya, T. (1998). Streptomycin mimics the cool temperature response in rice plants. J. Exp. Bot. 49: 221–227.[Abstract/Free Full Text]

Yoshioka, M., Uchida, S., Mori, H., Komayama, K., Ohira, S., Morita, N., Nakanishi, T., and Yamamoto, Y. (2006). Quality control of photosystem II: Cleavage of reaction center D1 protein in spinach thylakoids by FtsH protease under moderate heat stress. J. Biol. Chem. 281: 21660–21669.[Abstract/Free Full Text]

Yu, F., Fu, A., Aluru, M., Park, S., Xu, Y., Liu, H., Liu, X., Foudree, A., Nambogga, M., and Rodermel, S. (2007). Variegation mutants and mechanisms of chloroplast biogenesis. Plant Cell Environ. 30: 350–365.[CrossRef][Medline]

Yu, F., Park, S., and Rodermel, S.R. (2004). The Arabidopsis FtsH metalloprotease gene family: Interchangeability of subunits in chloroplast oligomeric complexes. Plant J. 37: 864–876.[CrossRef][Web of Science][Medline]

Yu, F., Park, S., and Rodermel, S.R. (2005). Functional redundancy of AtFtsH metalloproteases in thylakoid membrane complexes. Plant Physiol. 138: 1957–1966.[Abstract/Free Full Text]

Zaltsman, A., Feder, A., and Adam, Z. (2005a). Developmental and light effects on the accumulation of FtsH protease in Arabidopsis chloroplasts—Implications for thylakoid formation and photosystem II maintenance. Plant J. 42: 609–617.[CrossRef][Web of Science][Medline]

Zaltsman, A., Ori, N., and Adam, Z. (2005b). Two types of FtsH protease subunits are required for chloroplast biogenesis and photosystem II repair in Arabidopsis. Plant Cell 17: 2782–2790.[Abstract/Free Full Text]




This article has been cited by other articles:


Home page
 Plant Cell PhysiolHome page
W. Sakamoto, Y. Uno, Q. Zhang, E. Miura, Y. Kato, and Sodmergen
Arrested Differentiation of Proplastids into Chloroplasts in Variegated Leaves Characterized by Plastid Ultrastructure and Nucleoid Morphology
Plant Cell Physiol., December 1, 2009; 50(12): 2069 - 2083.
[Abstract] [Full Text] [PDF]

Home page
 Plant Physiol.Home page
Y. Kato, E. Miura, K. Ido, K. Ifuku, and W. Sakamoto
The Variegated Mutants Lacking Chloroplastic FtsHs Are Defective in D1 Degradation and Accumulate Reactive Oxygen Species
Plant Physiology, December 1, 2009; 151(4): 1790 - 1801.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. ProteomicsHome page
B. Zybailov, G. Friso, J. Kim, A. Rudella, V. R. Rodriguez, Y. Asakura, Q. Sun, and K. J. van Wijk
Large Scale Comparative Proteomics of a Chloroplast Clp Protease Mutant Reveals Folding Stress, Altered Protein Homeostasis, and Feedback Regulation of Metabolism
Mol. Cell. Proteomics, August 1, 2009; 8(8): 1789 - 1810.
[Abstract] [Full Text] [PDF]

Home page
 Plant CellHome page
E. Delannoy, M. Le Ret, E. Faivre-Nitschke, G. M. Estavillo, M. Bergdoll, N. L. Taylor, B. J. Pogson, I. Small, P. Imbault, and J. M. Gualberto
Arabidopsis tRNA Adenosine Deaminase Arginine Edits the Wobble Nucleotide of Chloroplast tRNAArg(ACG) and Is Essential for Efficient Chloroplast Translation
PLANT CELL, July 1, 2009; 21(7): 2058 - 2071.
[Abstract] [Full Text] [PDF]

Home page
 Plant CellHome page
J. Kim, A. Rudella, V. Ramirez Rodriguez, B. Zybailov, P. D. B. Olinares, and K. J. van Wijk
Subunits of the Plastid ClpPR Protease Complex Have Differential Contributions to Embryogenesis, Plastid Biogenesis, and Plant Development in Arabidopsis
PLANT CELL, June 1, 2009; 21(6): 1669 - 1692.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
20/7/1786    most recent
tpc.107.054965v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yu, F.
Right arrow Articles by Rodermel, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yu, F.
Right arrow Articles by Rodermel, S.
Agricola
Right arrow Articles by Yu, F.
Right arrow Articles by Rodermel, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
ASPB Publications THE PLANT CELL PLANT PHYSIOLOGY
Copyright © 2008 by the American Society of Plant Biologists