Review
Pentatricopeptide repeat proteins: a
socket set for organelle gene
expression
Christian Schmitz-Linneweber1 and Ian Small2
1
Institute of Biology, Humboldt University of Berlin, Chausseestr. 117, 10115 Berlin, Germany
2
Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley 6009, WA,
Australia
Pentatricopeptide repeat (PPR) proteins are RNA-bind-
ing proteins that are particularly prevalent in terrestrial
plants. Although the PPR protein family was only recog-
nized eight years ago, it is already clear that these
proteins have a range of essential functions in post-
transcriptional processes (including RNA editing, RNA
splicing, RNA cleavage and translation) within mito-
chondria and chloroplasts. Several PPR proteins have
been shown to act as fertility restorer genes in commer-
cially important cytoplasmic male sterility systems.
Here, we discuss several recent papers that cover their
evolutionary history and molecular mode of action. We
use these new data to propose hypotheses for their
physiological roles that could explain why PPR proteins
are so numerous in terrestrial plants.
What are pentatricopeptide repeat proteins?
The pentatricopeptide repeat (PPR) family was first recog-
nized from the Arabidopsis thaliana genome sequence [1],
although a few individual proteins had been characterized
previously. They are defined by the presence of a canonical
35-amino-acid motif [1], repeated in tandem up to 30 times.
Although no experimental structural information is avail-
able, they are predicted to comprise an array of a helices
[1], placing them in the ‘a-solenoid’ superfamily that
includes tetratricopeptide repeat (TPR) proteins, ankyrin
repeat proteins, HEAT domain proteins and Puf domain
RNA-binding proteins. Plant PPR proteins can be separ-
ated into two major subfamilies based on the nature of
their PPR motifs [2] and into several smaller subclasses
based on a series of characteristic C-terminal motifs
(Figure 1). Genetic, molecular and biochemical evidence
suggests that most PPR proteins have sequence-specific
RNA-binding activity, although the structural basis for
this is not known (reviewed in Ref. [3]). They have been
proposed to be molecular adaptors bringing RNA proces-
sing activities to target RNAs [2]. To date, all confirmed
physiological roles of PPR proteins are within mitochon-
dria or chloroplasts (reviewed in Refs [4,5]).
Here, we discuss the recent literature on these proteins
and highlight the most exciting recent developments.
Our current understanding is lacking in many areas, and
several promising future research directions are evident.
Corresponding author: Small, I. (iansmall@cyllene.uwa.edu.au).
1360-1385/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2008.10.001 Available online 12 November 2008
Genetic approaches to elucidating the functions of PPR
proteins
The rice (Oryza sativa) and Arabidopsis PPR families are
surprisingly similar, far more so than other large gene
families that have been studied in detail (the appearance
and expansion of the PPR family are discussed in Box 1).
For >80% of family members, clear orthologues are evident
in both species [6]. The low rates of gene loss or gene
duplication are consistent with strong selection pressure
on PPR genes and little redundancy between different
members of the family. This view is reinforced by the
strong phenotypes revealed in mutants lacking expression
of single PPR genes [2,7].
PPR mutants
Functional analysis of PPR genes has relied predominantly
on genetic tools. Most of what we know about the roles of
this protein family is based on single-gene studies inves-
tigating hypomorphic or – more often – null alleles of PPR
genes. The first PPR gene mutant was identified in Sac-
charomyces cerevisiae [8], but given the overwhelming
numbers of PPR genes in plants, it is not surprising that
‘green’ studies are now dominating the field. Following the
isolation of a transposon-induced null allele of the maize
(Zea mays) CHLOROPLAST RNA PROCESSING 1
(CRP1) gene [9], several dozen additional plant PPR gene
mutants have been described, most of them in Arabidopsis
thaliana (Table 1). The corresponding phenotypes are
diverse, and the defects are often surprisingly strong.
With few exceptions, PPR mutants affected in core
organellar functions are lethal, and many mutants isolated
thus far do not develop past the embryo stage [2,7,10,11].
Strong effects on embryo development are expected when
mitochondrial functions are compromised; for example, the
maize empty pericarp 4 (emp4) and the Arabidopsis orga-
nelle transcript processing 43 (otp43) PPR mutants are
both characterized by severely reduced germination rates
[12,13]. Less well understood is the impact of chloroplast-
localized PPR proteins on embryo development, because
Arabidopsis embryos can pass well beyond germination
without photosynthesis. Mutants defective in carbon fix-
ation, such as the Arabidopsis PPR mutants high chlor-
ophyll fluorescence 152 (hcf152) or otp51, usually die at the
seedling stage under autotrophic growth conditions
[14,15]. Therefore, a plastid function separate from photo-
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Figure 1. Schematic representation of plant PPR proteins. Rfo (restorer of fertility for Ogura male fertility in Brassica napus; [21]) is a typical P class PPR protein essentially
comprising only canonical 35-amino-acid PPR repeats. By contrast, the PLS class proteins CLB19 [38] and CRR2 [42] contain characteristic triplets of P, L (‘long’, generally 36
amino acids) and S (‘short’, generally 31 amino acids) motifs [2] and equally characteristic C-terminal domains. PLS class proteins were termed plant combinatorial and
modular proteins (PCMPs) in early literature [72]. The E (‘extended’) domain found in most PLS class proteins was previously divided into smaller motifs [72] (termed E and
E+ in Ref. [2]), but the published data do not support any functional distinctions between the known variants, so we refer to the entire region as the E domain. The C-
terminal DYW domain (named for its characteristic aspartate-tyrosine-tryptophan C-terminal tripeptide [2]) is only found in a subset of PLS class proteins.

synthesis must account for the observed block in embryo
development in many mutants defective in specific plastid
PPR proteins (Box 2).
Cytoplasmic male sterility
Cytoplasmic male sterility (CMS) is a failure to make pollen
by normally hermaphroditic plants owing to a genetic factor
carried by mitochondria [16]. CMS is widely used in plant
breeding for producing hybrid seed in species that are
otherwise self-compatible and, therefore, is of considerable
commercial interest. In most crop species, the hybrids pro-
duced will be required to set seed and, therefore, ideally
must be male fertile; to achieve this, the male parents in
these crosses carry nuclear ‘restorer’ (Rf) genes capable of
overriding the mitochondrial CMS-inducing gene. In most of
the CMS systems studied in detail, this mitochondrial gene
encodes a hydrophobic CMS-specific polypeptide, and the
corresponding Rf gene encodes a PPR protein that prevents
expression of the mitochondrial factor (Table 1). Intrigu-
ingly, most of these Rf genes seem to share common charac-
teristics, even from species as diverged as rice and brassicas.
With the exception of the Sorghum bicolor Rf1 gene [17],
they all encode P-class PPR proteins that are present among
small clusters of similar genes (many of which seem to be
pseudogenes [18–21]). These Rf clusters differ completely in
their evolutionary patterns from the bulk of PPR genes.
They show rapid changes in both dicots [21,22] and mono-
cots [23] with some evidence for diversifying selection [22].
The Arabidopsis (autogamous) and poplar (dioecious) gen-
omes contain similar clusters of Rf-like PPR genes [6], and
because neither plant has reproductive strategies where
restorer–CMS systems are likely to arise naturally, these
genes probably have other, so far unrecognized roles. Plant
mitochondrial genomes contain many transcribed but
apparently untranslated open reading frames (ORFs),
and we speculate that Rf-like PPR proteins might play
a part in preventing their expression. The Arabidopsis
Rf-homologous genes generate considerable numbers of
siRNAs, and many of their transcripts are cleaved by
gene-silencing pathways [24].
The exact molecular function of Rf proteins is currently
unknown. Their expression leads to a decrease in expres-
sion of the corresponding mitochondrial CMS factor [16],
but how this is achieved is not yet clear. By analogy with
other PPR proteins, Rf gene products are likely to bind
directly to mitochondrial transcripts encoding CMS fac-
tors, and there is some evidence for this [25]. Some Rf
factors change the profile of their target mitochondrial
transcripts (decreased stability or changes in RNA proces-
sing patterns, e.g. [26]), but they might function largely by
preventing translation [27].
More-detailed biochemical studies of Rf protein func-
tion are required to clarify their mode of action. The lack of
CMS in Arabidopsis has precluded genetic studies in this
model, but the identification of 20 or so Rf homologues
opens the door to testing other possible functions.
Sequence data on Arabidopsis and Brassica relatives will
be invaluable for understanding the evolution of the Rf
clusters. Sequence data from other plants might soon be
sufficient for testing the hypothesis that the monocot and
dicot Rf genes share a common ancestor distinct from
that of all other PPR genes. The realization that different
Rf genes are related (and likely to be clustered) will
accelerate the mapping and cloning of new examples
[28], making CMS systems more tractable in breeding
programs.
Molecular functions of PPR proteins
Rapid progress has been made recently in understanding
the functions of PPR proteins at the molecular level.
Genetic studies on PPR genes have left little doubt that
the majority of these proteins are involved in organellar
RNA metabolism. This widely accepted conclusion
should not hide the scarcity of biochemical data available
on PPR proteins. Many of the postulated activities of
PPR proteins still lack direct biochemical proof and thus
leave open the possibility that their roles are more
indirect than suspected and require additional, uniden-
tified factors. However, the most parsimonious expla-
nation for the observed functions of PPR proteins in
editing, splicing, stability and translation of various
transcripts is a direct interaction between each PPR
protein and a specific site in target RNAs (summarized
in Figure 2).

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Box 1. Origins and distribution of PPR proteins
The most striking observation at the time of the discovery of the PPR
family was the sheer numbers involved. The Arabidopsis thaliana
genome encodes 450 PPR proteins [2], and other terrestrial plant
genomes sequenced since encode even more (Figure I). Expressed
sequence tag (EST) data from a range of angiosperms suggest that PPR
genes are numerous in all terrestrial plants, including gymnosperms
and bryophytes. By contrast, the algal genomes sequenced so far
contain relatively few PPR genes, on a par with all other eukaryotes.
This has prompted several discussions on the mechanisms underlying
the massive expansion of the family in terrestrial plants [2,76]. A recent
comparison of the PPR sets from Arabidopsis, rice and the moss
Physcomitrella patens uncovered evidence from intron number and
positioning that suggests the more recent members of the family arose
by reverse transcription, implying retrotransposition as a probable
mechanism for sudden expansion [6].
The origins of the PPR motif and the C-terminal domains specific
to the terrestrial plant members of the family remain obscure. The
Figure I. Distribution of PPR genes among eukaryotes. The numbers of P class (yellow bars) and PLS class (green bars) PPR genes in a range of eukaryotes. The total
number of PPR genes in each organism is given on the right. The numbers are taken from Refs [2,6], apart from grape (Vitis vinifera) [73], poplar (Populus trichocarpa)
[74] and Chlamydomonas reinhardtii [75], which are based on analysis of the recently released genome sequences. It is probable that the numbers in grape and poplar
will be revised with improved coverage and assembly.
RNA-binding activity
Biochemical identification of in vivo RNA ligands by immu-
noprecipitation and subsequent analysis of bound RNAs
has been achieved for the maize plastid PPR proteins
CRP1, PPR4 and PPR5 and the Petunia hybrida mitochon-
drial Rf592 protein [25,29–31]. This technique is poorly
resolutive and only demonstrates association, rather than
physical interaction of a PPR protein with its target.
Detailed in vitro binding studies with genetically identified
target RNAs are only available for the Arabidopsis plastid
proteins HCF152 [32] and CHLORORESPIRATORY
REDUCTION 4 (CRR4) [33], rice mitochondrial Rf1 [27]
and maize PPR5 [34], the latter thus being the only protein
for which in vivo binding studies are matched by in vitro
studies. Additional PPR proteins have been demonstrated
to bind RNA in vitro, but no further attempts have been
made to demonstrate specificity [2,34,35].
Trends in Plant Science Vol.13 No.12
ubiquity of PPR genes in eukaryotes and the strict association with
organelle functions would suggest a mitochondrial origin, but no
mitochondrial genomes encode PPR proteins, nor do the bacterial
genomes thought to most closely resemble the ancestral endo-
symbiont. The Rhodobacter sphaeroides protein A3PM02 [77] and
the protein Q64EN5 from an uncultured deep-sea archaeon [78] are
the only ones from prokaryotes to show obvious homology to
eukaryotic PPR proteins. These isolated genes are probably
remnants of horizontal transfer events rather than an indication
of a prokaryotic origin of the whole family. A large amount of new
sequence data are expected shortly that should improve our
understanding of PPR protein distribution and evolution. Of
particular interest will be genome sequences from other bryo-
phytes, particularly those with either abundant RNA editing (e.g.
Selaginella moellendorffii) or no editing at all (e.g. Marchantia
polymorpha, recently chosen as a target for sequencing by the US
Department of Energy).
Together, these studies have shown that PPR proteins
attach to diverse RNA species with target sequences
located in 50 untranslated regions (UTRs) in the case
of CRP1 and CRR4 [30,33], introns in the case of PPR5
and PPR4 [29,31] and an intergenic spacer in the case of
Rf1 [27]. No commonalities in sequence motifs or sec-
ondary structures have been identified, so the basis for
RNA recognition by PPR proteins remains obscure.
Although it is generally assumed that the PPR motifs
form the RNA-binding domain, there is still little exper-
imental evidence for this idea. The only convincing evi-
dence comes from domain-swap experiments between
CRR21 and CRR4 that demonstrate that binding speci-
ficity is a function of the PPR tract and not of additional
domains [36]. Point mutagenesis of PPR domains and
eventually crystal structures are needed to fully answer
this question.
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Table 1. Mutants in PPR genes of autotrophic organismsa.

Species Function in RNA metabolism b Name c Refs
Maize (Zea mays) Translation [9,30,50]
RNA stability [31,34,62]
RNA splicing [31,34,29]
Unknown [12]
Rice (Oryza sativa) RNA cleavage [26,27]
RNA degradation [26]
Unknown [63]
Radish (Raphanus sativus) Decreased CMS-ORF protein level [20,21,64]
Arabidopsis thaliana Transcription [65]
Translation [66]
RNA stability [46]
RNA cleavage [14,42,67]
RNA splicing [13–15,67]
RNA editing [33,36–38]
Unknown [35,55,68–71]
Petunia hybrida Unknown [19,25]
Physcomitrella patens RNA splicing [45]
Chlamydomonas reinhardtii RNA stability [47,53]
a
Key to targeting (with a few exceptions experimentally verified): , cytoplasm; , mitochondria; black, nucleus (no plant PPR proteins are known to be localized in more
than one compartment); solid underline, in vivo binding to target verified experimentally; dashed underline, in vitro binding to target verified experimentally.
b
RNA cleavage: loss of an endonucleolytic cleavage event in mutants; RNA degradation: loss of RNA decay in the mutant; RNA stability: degradation of specific transcripts in
the mutant.
c
Multiple nominations possible.
d
Essential gene; i.e. null mutants do not reach maturity under auxotrophic conditions.
Abbreviations: dg1, delayed greening 1; emp4, empty pericarp 4; grp23, glutamin-rich protein 23; loi, lovastatin insensitive 1; loj, lateral organ junction; ptac2, plastid
transcriptionally active chromosome 2; Rfk1, restorer of fertility for Kosena male fertility in Brassica napus.

Specificity factors in RNA editing mRNA, which codes for a subunit of the chloroplast
The first demonstration that a PPR protein was involved in NAD dehydrogenase [37]. Subsequently, CRR21 was
RNA editing showed that CRR4 is required for editing of shown to be required for editing of a different site in ndhD
the start codon of the Arabidopsis chloroplast ndhD transcripts [36]. A third PPR protein, CHLOROPLAST

Figure 2. Molecular functions of chloroplast PPR proteins. This figure lists PPR protein functions that are well supported by genetic and biochemical experiments, including
binding data. (1) Translation. PPR proteins participate in translation initiation by binding to specific sequence elements in the 50 UTR of mRNAs. (2) Editing. Binding of PPR
proteins to short cis-elements immediately upstream of RNA editing sites is required for C-to-U processing. (3) Splicing. Sequence elements in group II introns of highly
convoluted unspliced precursor RNAs are entry sites for PPRs. This interaction is essential for splicing. (4) RNA stability. PPR proteins have a role in RNA cleavage and
stability. Binding of PPR proteins could recruit endonucleases and thus lead to RNA cleavage. Conversely, binding could prevent endonucleolytic cleavage by blocking
access of RNases, thus stabilizing the RNA. Possibly, binding occurs in the vicinity of secondary structure elements, such as hairpins, that are known targets for
endonucleases. Ultimately, PPR proteins guarantee production of a correctly processed, mature RNA that, in the case of mRNAs, is then subject to translation.

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Box 2. Why are so many mutations in PPR genes embryo-
lethal?
More than 30 mutants in plant PPR genes have been analysed so far
(Table 1, main text). Many of these genes are essential for plant
survival and are required to reach maturity (i.e. seed production).
Many other null mutations in Arabidopsis thaliana PPR genes lead
to early embryo abortion and thus non-viable seeds [2,7,10].
Interestingly, mutants in putative maize orthologues of two such
Arabidopsis embryo-essential PPR genes germinate normally
[29,31]. Mutations in maize PPR4 and PPR5 severely reduce plastid
translation [29,31], suggesting that differences in the coding
potential of maize and Arabidopsis plastid chromosomes could
account for the different phenotypes [31]. Tobacco plastids contain
four genes known to be essential for cell survival (reviewed in Ref.
[79]). Three of these, ycf1, ycf2 (functions unknown) and accD
(encoding a subunit of the acetyl-CoA carboxylase), are present in
Arabidopsis but absent from maize plastids. The failure to translate
one or more of these three genes is likely to be the reason for
embryo-lethality in Arabidopsis. An important experiment is to
determine whether AtPPR4 and AtPPR5 share conserved functions
with their putative maize orthologues.
Analysis of embryo-lethal PPR genes in Arabidopsis will require
the isolation of hypomorphic or conditional mutants; however, only
a few such mutants have been studied in seed plants [42,46]. More
thorough and successful applications of hypomorphic PPR mutants
have been reported in other organisms. In Chlamydomonas, an
allelic series of hypomorphic mutants helped to demonstrate that
the MCA1 protein is rate-limiting for the expression of cytochrome f,
a core subunit of the photosynthetic cytochrome b6f complex [47]. In
humans, patients with a C-to-T missense mutation in the leucine-
rich pentatricopeptide repeat cassette (LRPPRC) gene suffer from
reduced mitochondrial activity in the liver (Leigh syndrome French
Canadian variant [80]). In yeast, a detailed mutational analysis
demonstrated that PET309’s entire PPR tract is required for
translation of mitochondrial mRNAs and, in addition, identified
basic residues essential for translation of the target cox1 mRNA [51].
These studies should stimulate the use of similarly detailed
experiments in plants.
BIOGENESIS 19 (CLB19), is needed for editing of specific
sites in plastid clpP and rpoA transcripts, coding for sub-
units of a chloroplast protease (Clp) and the chloroplast
RNA polymerase, respectively [38]. All three are E-class
PPR proteins. CRR4 binds to the ndhD transcript that it
edits [33], and domain-swapping experiments between
CRR4 and CRR21 demonstrate that the PPR motifs are
responsible for the specificity of the editing reaction,
whereas the C-terminal E domain is required for the
editing reaction to occur [36]. It is generally thought that
the E domain is unlikely to carry the catalytic site and is
probably a protein–protein interaction domain that
recruits the as-yet-unknown editing enzyme [39]. The
DYW domain present in many PPR proteins has been
suggested as a candidate editing enzyme based on its
phylogenetic correlation with RNA editing in terrestrial
plants and the presence of invariant amino acids that
match those of the active site of known editing enzymes
from other organisms [6,40,41]; however, the only mutant
lacking a DYW-class PPR protein that has been described
shows an RNA cleavage defect rather than an editing
defect [42].
Further data from mutants in this class of proteins
should clarify their involvement (or not) in RNA editing,
and effort needs to be put into chasing interaction partners
of E-class PPR proteins to identify the editing enzyme(s)
that has/have escaped identification for almost 20 years.
Trends in Plant Science Vol.13 No.12
All of the data on PPR editing factors have come from work
on plastids, and it remains to be seen whether the large
number of similar PPR proteins predicted to be targeted to
mitochondria also specify editing sites. Editing specificity
factors are probably the best subjects for understanding
the basis for PPR recognition of specific RNA sequences
because the target site is precisely defined. Proteins such
as CLB19 [38] that recognize two non-identical sites will be
particularly useful in such studies.
Splicing, processing and RNA stability
Plant organellar genomes contain introns that fall into
either the group I or group II ribozyme classes. Although
theoretically self-splicing, it has been shown for most
chloroplast and some mitochondrial introns that
nuclear-encoded or, in some cases, organelle-encoded fac-
tors are required for proper excision from the precursor
RNA [43,44]. To date, seven PPR proteins have been
implicated directly or indirectly in organellar RNA spli-
cing. Yeast petite 309 (Pet309) is required for the accumu-
lation of intron-containing cytochrome c oxidase 1 (cox1)
mRNAs, but direct involvement in splicing remains to be
shown [8]. Moss (Physcomitrella patens) PPR531-11 has a
supporting role for splicing of the chloroplast clpP mRNA,
because residual amounts of spliced mRNAs are still
detectable in null mutants [45]. Similarly, HCF152
improves splicing of the petB-petD precursor RNA coding
for subunits of the cytochrome b6f complex [14] without
being essential. More stringent evidence for a direct role in
splicing has been presented for four other PPR proteins.
Maize PPR4 and PPR5 have been demonstrated to associ-
ate with high specificity in vivo with plastid introns in the
trans-spliced rps12 RNA (encoding chloroplast ribosomal
protein S12) and cis-spliced trnG(UCC) RNA (encoding
tRNAGly(UCC)), respectively [29,31]. Both proteins are
required for the accumulation of the spliced RNAs of their
cognate introns. The Arabidopsis plastid PPR protein,
OTP51, is essential for processing of intron 2 in the
hypothetical chloroplast open reading frame 3 (ycf3) mRNA
and seems to participate in splicing of other introns as well
[15]. In mitochondria, OTP43 is specifically required for
splicing of only the first of the four nad1 introns, another
trans-spliced intron [13].
Little is known about mechanistic aspects of PPR
proteins functioning in trans- or cis-splicing. Both PPR4
and OTP51 are non-canonical PPR proteins in that they
contain additional domains not generally found in PPR
proteins. PPR4 contains an RNA recognition motif (RRM),
whereas the OTP51 reading frame also encodes a LAGLI-
DADG endonuclease domain found in group I intron-
associated maturases. How these domains contribute to
splicing is unclear, although for PPR4, the protein might
make independent contacts via its separate RNA-binding
domains to both parts of the trans-spliced intron, thus
assuring close proximity of the intron-halves. Unfortu-
nately, the exact binding site has not yet been determined
for PPR4. A delimited binding site has only been deter-
mined for PPR5, which covers the exon binding site (EBS)
and a sequence elements within the intron [34]. These
elements are essential for the formation of the catalytically
active intron conformation. Possibly, PPR5 supports EBS
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and/or a function (e.g. by trapping a favourable RNA
secondary structure) and, thus, would be an integral part
in splice activity, but this remains to be proven – for
example by secondary structure analysis of the free versus
the PPR5-bound trnG(UCC) intron.
Much clearer than PPR5’s role in splicing is its import-
ance for stabilizing the trnG(UCC) precursor RNA that is
rapidly degraded upon loss of PPR5 [31]. RNA stabilization
is a function shared with other PPR proteins, such as
plastid MCA1 and PROTON GRADIENT REGULATION
3 (PGR3), as well as yeast mitochondrial PET309 [8,46,47].
An attractive hypothesis is that these proteins protect
nuclease-sensitive sites against one of the many plastid-
localized endonuclease species. Diametrically opposite to
this protective function are PPR proteins that promote
endonucleolytic cleavage, such as plastid CRR2, which is
required for processing of the ndhB-rps7 dicistronic pre-
cursor RNA in Arabidopsis [42], or plastid PPR531-11,
which supports cleavage between clpP and rps12 in Phys-
comitrella [45]. It is intriguing that PPR531-11, HCF152
and PPR5 all impact splicing and RNA cleavage of their
targets. Future studies need to address whether there is a
causal succession of these events mediated by PPR
proteins. Also, the identification of interacting RNases will
be important to understand the enhancing or inhibitory
action of PPR proteins on RNA cleavage and degradation.
Translational control
One of the open questions in plant organelle biology is how
translation initiation is achieved. Typical prokaryotic
Shine-Dalgarno sequences are lacking in organelle mRNAs
or show unusual spacing from the start codon [48,49]. It is
generally assumed that translation initiation proceeds by
mechanisms derived from the ancestral prokaryotic sys-
tem, but underlying factors have rarely been identified. An
exception is the chloroplast PPR protein CRP1, which is
required for translation of the petA and psaC transcripts,
which code for subunits of the cytochrome b6f complex and
photosystem I, respectively [9,50]. Its binding site in the
50 UTR points to a role in translation initiation, which is
supported by the lack of polysome association of target
mRNAs in crp1 null mutants [9]. It will be of interest to
analyse phylogenetic or structural relatives of CRP1 for a
role in translation of other organellar mRNAs. Outside the
plant PPR world, yeast Pet309 is also implicated in trans-
lation, although binding studies have not been undertaken
[51,52]. In the opposite direction, rice Rf1 prevents associ-
ation of orf79 transcripts with polysomes and presumably
blocks translation, because the ORF79 protein fails to
accumulate in Rf1 lines [27].
Regulation of gene expression
PPR proteins are required for a wide range of different
post-transcriptional processes in plant organelles, and the
lack of a particular PPR protein often leads to a severe
phenotype owing to a lack of expression of a specific
organelle gene (Table 1). They are therefore ideal candi-
dates to be gene-specific regulators of organelle gene
expression, and that many researchers believe that this
is the case explains much of the interest in them. However,
there is little hard evidence that any of the known PPR
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Trends in Plant Science Vol.13 No.12
proteins meaningfully regulates expression of an organelle
protein under physiological conditions. The best evidence
so far comes from Chlamydomonas reinhardtii, in which
the PPR protein MCA1 [53] has been shown to be a
regulator of the stability of chloroplast petA transcripts
[47]. MCA1 changes in abundance during changes in
nutrient availability, and these changes correlate with
changes in petA transcript levels and cytochrome f trans-
lation. Deliberately modifying MCA1 levels has the
expected effects on petA expression and is proof that
MCA1 can and does modulate cytochrome f expression
in a physiologically relevant manner [47]. It remains to
be demonstrated by similar experiments whether terres-
trial plant PPR proteins have similar regulatory roles.
PPR proteins might also affect nuclear gene expression.
Positional cloning of the GENOMES UNCOUPLED 1
(GUN1) gene implicated in plastid-to-nucleus signalling
showed it to be a PPR protein, although its mode of action is
not yet known [54,55].
Suppression of genome mutations
Although regulation of gene expression might be an
appealing task for PPR proteins, no regulatory role for
any terrestrial plant PPR protein has yet been demon-
strated. Thus, are there alternative explanations for the
size of the PPR family and its expansion in terrestrial
plants? Two considerations about the evolution of organel-
lar genomes in plants [56] are salient here.
Firstly, as endosymbionts with small, asexual, non-
recombining populations, both mitochondria and chloro-
plasts are expected to accumulate mildly deleterious
mutations; this phenomenon is well known from more
recent endosymbiotic events and has been termed Muller’s
ratchet [57,58].
Secondly, plant organellar genomes accumulate
mutations less frequently than do plant nuclear genomes
and more slowly than do metazoan mitochondrial genomes
[57]. Consequently, deleterious organellar mutations in
plants can be suppressed by the more rapidly evolving
nuclear genome, for example by import of ‘repair’ factors.
PPR proteins can be considered as factors that alleviate
genome mutations at the RNA level, for example by revert-
ing T-to-C mutations (editing sites), by restoring intron
structure or by blocking expression of recombined open
reading frames. If this view of PPR protein function is
correct, deletion of otherwise essential PPR genes should
be possible after artificial repair of the organelle genome to
remove the necessity for the relevant RNA-processing step.
For example, correction of an essential editing site in the
plastid atpA gene did not lead to any visible phenotype, at
least under laboratory conditions [59], implying that the
editing event is needed to suppress a genome error rather
than to regulate atpA expression.
Conclusions
There is no longer any doubt that the PPR protein family has
acquired many essential roles in organelle gene expression
in plants. A complete catalogue of all of the processes in
which they are involved will take considerable work, but the
genomics tools are now available to proceed rapidly from
mutant phenotype to molecular function [2,60,61].
Review
However, important outstanding questions remain:
firstly, how do PPR proteins recognize such a vast array
of RNA targets with such specificity? The PPR family is the
largest RNA-binding protein family for which no struc-
tures are known; a single structure of a PPR protein
complexed with an RNA ligand would be a huge step
forward. An understanding of RNA recognition by PPR
proteins will reveal whether there is a PPR motif ‘code’ that
can be read to predict target sequences. It might even
become possible to engineer PPR proteins to bind new
targets, thus opening up potential biotechnological appli-
cations where RNA binding specificity is desired.
Secondly, do the huge numbers of PPR proteins provide
terrestrial plants with unparalleled regulatory control over
organelle gene expression, or are they merely a curious,
historical accident? Synthetic biology approaches will reveal
which of these interpretations is the more accurate. Engin-
eering organelle genomes to remove the necessity to edit,
splice or process particular mRNAs is feasible, but will such
experiments produce fitter organelles by removing wasteful
RNA repair processes, or will they produce sick plants
unable to correctly regulate their gene expression?
Acknowledgements
This research was supported by Australian Research Council Centre of
Excellence grant CE0561495. I.D.S. is supported as a WA State Premier’s
Fellow. C.S.L. is supported as an Emmy-Noether young investigator by
the Deutsche Forschungsgemeinschaft.
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