Pentatricopeptide repeat proteins and their emerging roles in plants

Plant Physiology and Biochemistry 45 (2007) 521e534 www.elsevier.com/locate/plaphy

Review

Pentatricopeptide repeat proteins and their emerging roles in plants D. Saha, A.M. Prasad, R. Srinivasan* National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi 110012, India Received 6 July 2006; accepted 21 March 2007 Available online 24 March 2007

Abstract Several protein families with tandem repeat motifs play a very important role in plant development and defense. The pentatricopeptide repeat (PPR) protein family, one of the largest families, is the most perplexing one in plants. PPR proteins have been implicated in many crucial functions broadly involving organelle biogenesis and plant development. PPR motifs are degenerate motifs, each with 35-amino-acid sequences and are present in tandem arrays of 2e27 repeats per protein. Although PPR proteins are found in other eukaryotes, their large number is probably required in plants to meet the specific needs of organellar gene expression. The repeats of PPR proteins form a superhelical structure to bind a specific ligand, probably a single-stranded RNA molecule, and modulate its expression. Functional studies on different PPR proteins have revealed their role in organellar RNA processing, fertility restoration in CMS plants, embryogenesis, and plant development. Functional genomic techniques can help identify the diverse roles of the PPR family of proteins in nucleus-organelle interaction and in plant development. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Chloroplast; Cytoplasmic male sterility; Mitochondria; Plant development; PPR proteins; Fertility restoration; RNA processing

1. Introduction Whole-genome sequencing projects of many eukaryotic organisms have identified several major gene families, most of whose functions cannot be ascribed or validated. Therefore, a major challenge of the post-genome sequencing task is to discover the functions of these genes. Sequencing of the model plant Arabidopsis thaliana genome and the exhaustive bioinformatic analysis of organelle-targeted proteins have identified a large gene family (more than 450 members) that encodes proteins containing pentatricopeptide repeats (PPRs). The PPR protein family is characterized by the signature motif of a degenerate 35-amino-acid repeat often arranged in tandem arrays of 2e27 repeats per peptide [66,96]. Many PPR proteins are predicted to be targeted to either the mitochondria

Abbreviations: CMS, cytoplasmic male sterility; GFP, green fluorescent protein; LOJ, lateral organ junction; ORF, open reading frame; PPR, pentatriocopeptide repeat; Rf, restoration of fertility; T-DNA, transferred DNA; TPR, tetratricopeptide repeat; UTR, untranslated region. * Corresponding author. Tel.: þ91 11 2584 1787x222; fax: þ91 11 2584 3984. E-mail address: [email protected] (R. Srinivasan). 0981-9428/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2007.03.026

or the chloroplast [66]. Despite extensive sequence analysis, the functions of several Arabidopsis proteins remain unidentified and PPR proteins constitute a considerable portion (about 10%) of these proteins [66]. Because most of these proteins are trafficked to the mitochondria and chloroplast, elucidating the function of PPR genes can reveal many important facets of the interaction between the nucleus and organelles in the plants. Structurally, PPR motifs closely resemble another repeat motif known as the tetratricopeptide repeat (TPR), generally found in eukaryotic proteins, especially yeast and Drosophila, and implicated in mediating proteineprotein interactions [25]. Because of the close resemblance, the term pentatricopeptide repeat was derived from the tetratricopeptide repeat [96]. However, PPR proteins differ from TPR proteins in some structural aspects (Table 1). Several proteins, having complex and variable arrangements of PPR motifs in different combinations, constitute a distinct class of PPR proteins, commonly known as plant combinatorial and modular proteins (PCMPs), and are evolved earlier than classical PPR motifs [66,87]. Unlike TPR-containing proteins, PPR proteins are sequence-specific RNA- or DNA-binding proteins [46,56,68,78,79,91,92,103] probably involved in modulating organellar gene expression by processing

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522 Table 1 PPR and TPR proteins at a glance Similarities

Presence of degenerate helical tandem repeat motifs Each repeat consists of anti parallel a helix (A and B) Repeat units form a super helix to bind biomolecules Conserved tyrosine residue helps in intra helix packing

Dissimilarities

Pentatricopeptide repeat Predominant in plants Generally absent in prokaryotes PPR proteins interact mainly with RNA (DNA in few cases) Number of repeats are more (2e27) as compared to TPR 35 amino acid repeats Side chains of amino acids in the central groove are exclusively hydrophilic PPR protein binds to a single target molecule (single stranded RNA)

and stabilizing mRNA [11,35], regulating translation [91], or both [108]. A comprehensive review by Andres et al. [6] discusses the diverse possible roles of PPR proteins in organellar gene expression. In plants, the association of PPR proteins with an RNA molecule or the processing of transcripts has been observed in PPR-related mutants from Chlamydomonas [65], Arabidopsis [42,74,108], and maize [107]. Many plant PPR genes that have been ascribed possible functions thus far are involved in either male fertility restoration through modification or silencing of cytotoxic mitochondrial transcripts [3,12,14,25,49,52,53,105], or post-transcriptional modulation of plastid gene expression [11,35,54,56,74,108]. PPR genes in Arabidopsis play an essential role in plant embryogenesis [21,27] and other plant developmental processes [81,83]. Table 2 lists some PPR genes and their suggested functions. In most cases, every member of the PPR protein family is suggested to bind to a specific organellar RNA and recruit general factors for its processing, maturation, and translation, thus regulating its expression [54,55,66,79,91,108]. In this review, we briefly discuss the structural features of PPR proteins and summarize the information currently available on PPR proteins and the variety of functions identified for some PPR genes in plants. 2. Structural organization of PPR proteins 2.1. Classical PPR motifs A typical PPR-containing protein consists of a variablelength organelle-targeting sequence at the NH2-terminal end and 2e27 PPR repeats in a tandem array (Fig. 1A). PPR proteins are similar in structure to proteins with TPR repeats [24], consisting of a pair of antiparallel a helices: helices A and B [96]. Tandemly organized repeats in TPR proteins form a central groove that is predicted to serve as a macromolecular binding site [24]. Based on their structural resemblance with TPR motifs, PPR repeat motifs are predicted to form a structure that serves as a binding site for a singlestranded RNA molecule. Structurally, helix A is exposed in the groove of the superhelix and helix B is on the outside.

Tetratricopeptide repeat Mostly abundant in animals and lower plants Present in prokaryotes TPR interacts mainly with other proteins Less number of repeats (3e16) as compared to PPR 34 amino acid repeat units Amino acids in the central groove vary considerably TPR proteins may bind to multiple target proteins forming a complex

The conserved tyrosine molecules are probably involved in interhelix packaging in both PPR and TPR proteins [96]. Despite the striking structural similarities between TPR and PPR proteins, there are significant differences between them, especially at the end of helix B, the connecting loop between the repeat motifs, and throughout most of helix A (Table 1). The amino acid residues projecting inside the tunnel vary considerably in TPR proteins, reflecting the variety of ligands bound, whereas the side chains of amino acids lining the central cavity of PPR proteins are strictly hydrophilic. Predicted models of several PPR-containing proteins show that the bottom of this groove is positively charged, thereby facilitating the binding of negatively charged nucleotides of RNA molecules. PPR-containing proteins have, on average, twice as many repeats as those in TPR proteins, suggesting multiple or extended room for ligand binding [96]. Apart from the predominant PPR repeat motifs, several other variable motifs or domains have been found in various PPR proteins. Fig. 1B provides a schematic representation of PPR repeats and some additional domains detected by Pfam v.21.0 [33] in some PPR proteins of Arabidopsis. 2.1.1. PPR-like motifs in plant combinatorial and modular proteins (PCMPs) Classical PPR repeats (designated as P-motifs) are arranged in tandem, but they may be placed either without any gaps or with a regular gap of approximately 70 amino acids. Bioinformatic alignments of these interrepeat regions have shown two new PPR-like motifs with 31 and 35e36 amino acids in some PPR proteins. These additional motifs are designated PPR-S (for short) and PPR-L (for long) [66]. Thus, on the basis of length and arrangement of repeated motifs, PPR proteins are broadly grouped into two subfamilies, classical PPR proteins with P-motifs only and plant combinatorial and modular proteins (PCMPs) with repeated blocks of P-L-S motifs [8,87] (Table 3). The complex and combinatorial structural arrangement of P-L-S repeats might explain the varied ligand-binding specificity of individual PCMPs [87]. Studies on PCMPs are still in their infancy, and further investigations will reveal the mechanism of their interaction with other RNA-interacting proteins and the role played by combinatorial motifs.

Table 2 A list of plant pentatricopeptide repeat protein genes and their suggested functions (in alphabetical order) Targeting organelle/(RNA)a Suggested functions

Effect of mutated gene

P-subfamily

Mitochondria/(not known)

Embryo development

P-subfamily P-subfamily

Mitochondria/(not known) Chloroplast/( petA, psaC )

9

PCMP-H

Chloroplast/(rps7/ndhB)

Circadian rhythmic expression Processing of the petD mRNA and translation of the chloroplast petA and petD mRNAs RNA splicing between rps7 and ndhB by recruiting endonuclease

Arrested embryo growth during [51] globular to heart transition Not studied [81] Loss of the cytochrome b6f complex [11,35,91] and a reduction in photosystem I proteins

Arabidopsis

11

PCMP-E

Chloroplast/(ndhD)

Arabidopsis Arabidopsis

14 9

HCF152 (At3g09650) Arabidopsis

12

PCMP P- subfamily with C-terminal WQQ domain P-subfamily

Chloroplast/(not known) Nuclear/(RNA polymerase II, subunit III) Chloroplast/( psbH/petB)

LOJ (At2g39230)

Arabidopsis

19

P-subfamily

Mitochondria/(not known)

OsPPR1

Rice

11

P-subfamily

Chloroplast/(not known)

Processing and /or stabilization of polycistronic ( psbB-psbT-psbH-petB-petD) chloroplast transcripts Lateral organ development and boundary formation Chloroplast biogenesis

P67 (At4g16390)

2

P-subfamily

Chloroplast/(not known)

Processing or the translation of cpRNAs

PGR3 (At4g31850)

Arabidopsis and radish Arabidopsis

27

P-subfamily

Chloroplast/( petL/ndH )

Stabilization of the primary tricistronic transcript of the petL operon

PPR2 PPR4

Maize Maize

11 16

P-subfamily P-subfamily

AtPPR4 (At5g04810) Rf

Arabidopsis Petunia

16 14

P-subfamily P-subfamily

Chloroplast/(not known) Chloroplast/(1st intron of rps12 pre-mRNA) Chloroplast/(not known) Mitochondria/( pcf )

Rf1a

Rice

18

P-subfamily

Mitochondria/(atp6/orf79)

Rf1b

Rice

11

P-subfamily

Mitochondria/(atp6/orf79)

Rf1(PPR13) Rfo

Sorghum 14 Radish (Ogura) 16

PCMP-E P-subfamily

Mitochondria/(not known) Mitochondria/(orf138)

Rfk1

Kosena radish

P-subfamily

Mitochondria/(orf125)

Involved in ribosome accumulation in plastids Trans-splicing of rps12 RNA and ribosome biogenesis in plastids Plastid ribosome biogenesis Fertility restorer, alter the expression of CMS-associated genes, decrease the accumulation of CMS-associated PCF protein Fertility restorer, alter the expression of CMS-associated genes, by endonucleolytic cleavage decrease the accumulation of atp6 RNA, additional role in RNA editing Fertility restorer by post transcriptional degradation of CMS related transcript Fertility restoration Fertility restoration by decreasing the CMS related protein Fertility restoration by decreasing the CMS related protein

Plant

At1g53330

Arabidopsis

9

AtC401 (At5g21222) CRP1

Arabidopsis Maize

12 14

CRR2 (At3g46790)

Arabidopsis

CRR4 (At2g45350) EMB175(At5g03800) GRP23 (At1g10270)

16

Target RNA either experimentally verified or predicted from mutant analysis.

RNA editing by acting as transacting factor to recruit C-deaminase Early embryo development in plants Transcriptional regulator at the early stage of embryo development

References

Loss of post illumination chlorophyll fluorescence upon imaging with chlorophyll fluorometer Lack of post-illumination increase in chlorophyll fluorescence Embryo-lethal phenotype Defective embryos

[42]

Impaired petB intron splicing or stabilization of splicing products

[74,78,79]

No observable phenotype

[83]

Chlorophyll deficiency, albinism and lethality Not studied

[37]

Aberrant chlorophyll fluorescence and affect the accumulation of petL operon RNA. Albino or ivory leaves Ivory leaf pigmentation

[108]

Embryo lethality Not studied

[92] [12]

Not studied

[3,49,53,105]

Not studied

[3,105]

Not studied Not studied

[50] [14,25]

Not studied

[52]

[54,82,93] [21] [27]

[56]

[107] [92]

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a

PPR PPR sub repeats family

D. Saha et al. / Plant Physiology and Biochemistry 45 (2007) 521e534

PPR genes

D. Saha et al. / Plant Physiology and Biochemistry 45 (2007) 521e534

524

A

COO-

H2N

Variable length organelle targeting sequence

B

2-27 PPR repeats in tandem 35 amino acid per repeat unit

C terminal optional motif

At1g53330 (471 aa) At5g21222 (AtC401) (831 aa) At3g46790 (CRR2) (657 aa) At2g45350 (CRR4) (606 aa) At5g03800 (EMB175) (896 aa) At1g10270 (GRP23) Gln rich WQQ

bZIP

(913 aa)

AT3G09650 (HCF152) (778 aa) AT2G39230 (LOJ) (867 aa) At4g16390 (P67) SMR

(688 aa)

At4g31850 (PGR3)

(1112 aa)

At5g04810 (AtPPR4) RRM

(952 aa)

Pkinase: Serine threonine protein kinase catalytic domain (S_TKc); bZIP: basic Leucine zipper; Gln: Glutamine; SMR: Small MutS Related; RRM: RNA recognition motif;

Fig. 1. (A) A typical structural arrangement of a pentatricopeptide repeat protein. (B) Schematic diagram showing PPR motif distribution in a few PPR proteins from Arabidopsis thaliana as obtained from Pfam v21.0 [32] (figures according to the scale of amino acid lengths). Tandem PPR motifs are represented as green and red boxes. Bars and ovals represent motifs other than PPR. Only the identified domains were mentioned. Total number of PPR motifs of individual PPR proteins may vary with different motif prediction tools. The exact PPR motif numbers as reported against concerned PPR proteins are provided in Table 2. (For interpretation of the references to colour in figure legends, the reader is refered to the web version of this article).

Table 3 Differences between classical PPR proteins and plant combinatorial and modular proteins (PCMPs) Sl. no.

Classical PPR proteins

Plant combinatorial and modular proteins (PCMPs)

1

Arrangement of pentatricopeptide repeat motifs (P-motifs) is simple and tandem P-repeat motifs are placed immediately adjacent to each other

Complex arrangement of repeat motifs in different combinations P-motifs are separated from each other by L (long) and S (short) motifs together as P-L-S- blocks PCMPs usually consist of C-terminal motifs like E, Eþ and DYW PCMPs are found only in land plants indicating its evolution after classical PPR proteins

2

3

4

P-motifs can occur singly without any carboxyl terminal motif Classical PPR proteins with only P-motifs are prevalent in plants, fungi and animals

2.1.2. C-terminal motifs The C-terminal ends of PPR proteins are variable and consist of three different optional motifs, E, Eþ, and DYW, which bear no sequence similarity with actual PPR motifs. These three motifs are present in PPR proteins and PCMPs only and not in any other protein of Arabidopsis (except At1g47580, which has a single DYW motif) [66]. The E and Eþ motifs are characterized by their degeneracy, whereas the DYW motif has a high amino acid conservation, particularly the Cys and His residues [8]. The three C-terminal motifs always occur in a single stretch and always appear in the typical conserved order of E, Eþ, and DYW; that is, Eþ-containing proteins must be preceded by the E motif and the DYW motif by E and Eþ motifs [66]. The occurrence of C-terminal motif is optional in classical PPR proteins but is usually associated with PCMPs and has been implicated in the recruitment of catalytic factors for RNA processing [87].

D. Saha et al. / Plant Physiology and Biochemistry 45 (2007) 521e534

3. Localization of PPR proteins in cell organelles Most PPR proteins contain organelle-targeting sequences to either the mitochondria or chloroplast [66,96]. The subcellular localization of these proteins is primarily studied by software tools with computational algorithms to identify the probabilities of targeting signals from amino acid sequences (Table 4). Experimentally, the subcellular localization of PPR proteins is determined by visualizing the expression of a reporter gene such as GFP or other organelle-specific visual markers fused to the ORFs of PPR proteins and introduced into plant cells for a transient or stable expression [37,66,82]. The transient expression of epitope-tagged PPR proteins along with the GFP reporter gene and western blotting have been used to detect the subcellular localization of P67 (At4g16390) in chloroplasts of Nicotiana plumbaginifolia [56]. Screening mutants of PPR proteins may help identify the organellar localization of the protein. For example, chlorophyll fluorescence imaging helps detect an impaired gene product in the chloroplast, which is followed by identifying the gene responsible, as shown in CRR4 [54] and PGR3 (At4g31850) [108]. The more recently developed microarray-assisted RNA immunoprecipitation [91,92] can help establish the association of a PPR protein with a specific organellar transcript and identify its working location. 4. Distribution of PPR protein genes in plants Although PPR proteins have been reported in all eukaryotes, their numbers are very high in plants as compared with other eukaryotes. For example, Drosophila and C. elegans each contain two PPR proteins, whereas the Arabidopsis genome contains more than 450 [66,87] and rice about 655 [65] PPR proteins, of which 198 members in Arabidopsis and 229 in rice belong to the PCMP subfamily [87]. Several other plant species such as maize [22,107], radish [14,25,52], Petunia [12], and sorghum [50] contain several PPR protein genes. The presence of PPR genes in lower plants such as Physcomitrella patens, a land moss [43], trypanosomes [38,71,77], and the red alga Cyanidiodschyzon meroale [72] suggests that these proteins possibly increased around the period of evolution of land plants. The most widely studied PPR proteins are from A. thaliana and are distributed in almost all the five chromosomes. In the long arm of chromosome 1, an approximately 1-Mb region is the most populated wherein 19 PPR genes and probably

525

several PPR pseudogenes reside in a cluster. Several PPR genes from this cluster are closely related to other PPR genes cloned from radish, Petunia, and rice coding for the CMS restorer [66]. One interesting feature of PPR-coding genes is that most are devoid of introns [66], in contrast to other Arabidopsis genes containing five introns on average [7]. As a result, PPR genes are usually short (average size 2.0 kb) despite their coding for a large protein with more than 650 amino acids [66]. The absence of intronic sequences from the PPR genes of Arabidopsis raises an important question about the evolutionary origin of these proteins in plants. One hypothesis suggests that in early plant evolution PPR genes might have multiplied through RNA intermediates; that is, they were transcribed into RNA, then reverse-transcribed to DNA and reinserted to the genome, resulting in gene duplication and loss of introns in many of these genes [5,58]. 5. PPR gene expression in plants A global expression profiling of PPR genes in Arabidopsis revealed that 40e50% of PPR genes are expressed and the products mostly localized into subcellular compartments such as mitochondria and chloroplasts. The overall expression of the PPR family of genes in Arabidopsis is very low [66]. This could be because the expression analysis was carried out in a few plant organs only, such as leaves and flowers, and not at all stages of plant growth. However, reports of localized expression of PPR genes in some plant parts point to their specific role in different plant tissues or cells. For example, there were higher levels of expression of the PPR gene P67 (At4g16390) in leaves and flowers of A. thaliana than in stem and flower buds. P67 was not expressed in roots [56]. In Petunia, the restorer gene Rf-PPR592 was expressed in floral buds only [12]. The rice fertility restorer gene Rf-1 has been shown to express in panicles during the development of pollen, at the booting stage, and in green leaves [3]. Furthermore, the atp6/orf79 transcript was processed in the presence of the Rf-1 gene in a rice callus, indicating its expression in not only a panicle containing immature pollen but also vegetative organs such as calli [3]. Reverse-transcription polymerase chain reaction (RT-PCR) analysis of the OsPPR1 transcript in different plant parts of rice revealed a preferential expression in leaves [37]. A 246-bp promoter of the PPR gene AtC401 (At5g21222) conferred a circadian rhythmic expression in cotyledons of a 5-day-old transgenic Arabidopsis seedling, indicating that the PPR gene is regulated by a circadian rhythm

Table 4 A list of important software tools for predicting sub-cellular localization of PPR proteins Sl. no.

Software tools

Prediction of organelles

References

URLs

1 2 3

PCLR v0.9 MITOPROT Predotar v1.03

[90] [18] [97]

http://andrewschein.com/pclr/ http://mips.gsf.de/cgi-bin/proj/medgen/mitofilter http://urgi.infobiogen.fr/predotar/predotar.html

4

TargetP 1.1

[29]

http://www.cbs.dtu.dk/services/TargetP/

5

ChloroP 1.1

Chloroplast Mitochondria Chloroplast, mitochondria and endoplasmic reticulum Predicts the subcellular location of eukaryotic proteins Chloroplast

[30]

http://www.cbs.dtu.dk/services/ChloroP/

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[81]. The Lateral Organ Junction (LOJ ) gene (At2g39230) of Arabidopsis thaliana identified in our laboratory codes for a PPR protein [83]. The upstream sequences of the LOJ gene directed the expression of the reporter gene GUS in all lateral organ junctions (LOJs). Detailed expression analysis of the LOJ transcript by RT-PCR in the loj mutant and wildtype Arabidopsis thaliana established a compartmentalized expression pattern of the gene in the LOJs [83]. Although these results are indicative only, further studies on the expression of PPR genes in specific tissue and cell types will help elucidate the specific role of PPR genes in regulating plant development. 6. Mutants of PPR genes in Arabidopsis Insertion mutants are valuable resources to obtain information about the function of a gene known by its phenotype [86]. Several T-DNA insertion mutants of PPR genes in Arabidopsis have been generated and homozygous mutants characterized genetically [21,27,51,74,83]. Many exhibit an embryo-lethal phenotype [21,27,51]. Phenotypes exhibited by other mutants include dwarf stature and reduced fertility [66]. Several embryo-defective mutants obtained from the Arabidopsis seed genes project contained T-DNA insertions in at least 17 PPR genes [104]. The T-DNA and Ac/Ds transposon generated knockout mutants of Arabidopsis emb175 (At5g03800), the CB_1265 mutant line (At1g53330) and grp23 (At1g10270) exhibited defects in embryogenesis and patterning of embryo morphology, resulting in embryo lethality [21,27,51]. Such results suggest that some PPR genes play an essential role in plant embryo development. Besides embryo-defective mutations, other T-DNA insertions in PPR genes show phenotypes associated with chloroplast RNA processing and faulty gene expression. An Arabidopsis mutant in the PPR gene At3g09650, designated as high-chlorophyll-fluorescence (hcf152-1), shows a defect in petB (photosynthetic electron transport) and psbH mRNA processing in the chloroplast, thereby resulting in reduced levels of the cytochrome b6f complex [74]. The loj mutant identified by us carried a T-DNA insertion in the 50 upstream region of a locus At2g39230, which codes for a PPR protein of unknown function. The mutation resulted in the loss of transcript, but no obvious phenotype was discernable. However, reporter gene expression and gene expression analysis suggested the association of the loj gene with the LOJs and shoot apical meristems [83]. Although at present only a few mutants available in PPR genes have been characterized completely, they indicate the involvement of PPR genes in a variety of functions in plant development. 7. Emerging roles of PPR proteins in plants 7.1. PPR proteins and RNA processing Transcription in plant cells occurs in three distinct subcellular compartments: nucleus, mitochondria, and chloroplast. Nuclear-encoded mRNAs are transcribed into large precursors and processed into mature mRNAs in several steps before

being exported to the cytoplasm. Throughout their maturation pathway mRNAs are stably associated with ribonucleoprotein (RNP) complexes [32,55]. The regulation involved in RNA processing and translation governs both nuclear and organellar gene expression. Many genetic and biochemical studies have revealed the complexity and diversity of RNA-protein interactions. Several classes of RNA-binding proteins have been identified based on conserved RNA-binding motifs [16,28]. In Arabidopsis, more than 200 proteins with RNA-binding motifs have been identified [63], most corresponding to the RRM and K-homology (KH) class [4]. Others include RNA helicases [9,64], poly A-binding proteins (PABP), glycine-rich proteins (GRP) [4], and the flowering time control gene in Arabidopsis (FCA) [67,85]. Although, no natural RNA substrates have been identified for most of these proteins, their interaction with specific RNA ligands possibly influences the fate of the RNA and further downstream processes [4]. PPR proteins are RNA-binding proteins [56,66,78,79,91,92], regulating the RNA processing, maturation, and translation. At the physiological level, PPR proteins have been shown to play an important role in several developmental processes in plants (Fig. 2). 7.1.1. PPR proteins in chloroplast RNA processing In land plants, chloroplast gene expression is an independent process induced during the development of photosystem components. The chloroplast genome of vascular plants encodes more than 120 genes usually transcribed in a polycistronic message, which needs to undergo extensive processing during gene expression [100]. In plants, several nuclearencoded RNA-binding proteins take part in chloroplast gene expression by processes such as transcription, RNA editing, RNA splicing, RNA degradation, and translation [10]. PPR proteins might be the major family involved in crucial functions of the chloroplast gene expression process. One of the earliest evidences of PPR proteins regulating chloroplast mRNA is that of the maize nuclear gene crp1, which is required for the processing and accumulation of petB and petD mRNAs from a polycistronic precursor. The CRP1 protein also activates the translation of another chloroplast mRNA petA [11,35]. These mRNAs were absent in crp1 mutants, which resulted in the loss of the cytochrome b6f complex [35], one of the four major multiprotein complexes in the chloroplast. The other three complexes involved in photosynthetic electron transfer and ATP synthesis are the photosystem I, photosystem II, and the ATP synthase [17]. The mutant crp1 exhibited a loss of psaC mRNA and photosystem I proteins, indicating the role of CRP1 in translation of the psaC mRNA [11]. CRP1 is a member of PPR protein family with 14 tandem PPR motifs [34,35,91]. Initially the observation that CRP1, a component of a multisubunit protein complex, was not associated with ribosomes led to the conclusion that CRP1 indirectly and independently influences the translation of petD and petA mRNA. This conclusion was further supported by the observations that CRP1 shares a strong homology with the PET309 of yeast [69,70] and the CYA-5 of Neurospora [19], which regulate translation and metabolism

D. Saha et al. / Plant Physiology and Biochemistry 45 (2007) 521e534

PPR encoding gene

Fertility restoration in CMS plants Post transcriptional processing or degradation of RNA

PPR mRNA

Nucleus

?

N- terminal organelle targeting sequence

Organelle trafficking

PPR repeats arranged in superhelix protein structure

?

Co factor

?

Embryogenesis

Plant Development

RNA processing Organelle biogenesis

? PPR protein bound to specific RNA

527

Translation

? RNA editing

C-U

Chloroplast or mitochondria

Fig. 2. A schematic diagram depicting major functions of PPR proteins in plants.

of mitochondrial cox1 mRNA, and thus might function as CYA-5 does [35]. Later, RNA immunoprecipitation and chip hybridization (RIP-chip) assay demonstrated that CRP1 is directly associated with petA and psaC mRNAs in vivo and activates specifically the translation of both these mRNAs [91] (Section 7.2.1). Another protein P67 identified from both radish and Arabidopsis (At4g16390) has two PPR motifs and has been implicated in the formation of machinery for the processing and translation of specific chloroplast mRNAs [56]. The Arabidopsis gene HCF152 (At3g09650) encodes an 80 kDa soluble protein with 12 putative PPR motifs targeted to the chloroplast and is probably involved in the maturation of chloroplast transcripts. The protein HCF152 is an RNA-binding protein that binds to the introneexon junctions of petB mRNA and the region between psbH and petB with very high affinity. On binding, it is probably involved in splicing and stabilization of the multicistronic psbB-psbT-psbH-petB-petD transcript. Mutants of HCF152 exhibit high chlorophyll fluorescence because of the reduced levels of the photosynthetic b6f complex [74,78,79]. Similar to HCF152, another nucleus-encoded factor PGR3 with 27 PPR repeats was identified from the EMSgenerated proton gradient regulation3 ( pgr3) (At4g31850) mutant of Arabidopsis. The allelic pgr3 mutants produced inconsistent chlorophyll fluorescence because of a defect in the chlorophyll gene expression process and a consequent reduction in levels of the b6f complex. Further molecular analysis of pgr3-1 and pgr3-2 mutants revealed a defect in the accumulation of petL mRNA and reduced levels of the b6f complex. Unlike the mechanism of action of CRP1 and HCF152, PGR3 was speculated to function not by processing but by stabilizing and activating the petL mRNA translation, thereby regulating multiple and specific chloroplast gene expression

processes [108]. The rice PPR gene OsPPR1 with 11 PPR repeats is involved in the processing of chloroplast RNAs required for early biogenesis of plastids [37]. The PPR protein CRR4 (At2g45350) is essential for RNA editing in ndhD in chloroplasts of Arabidopsis. CRR4 belongs to the PCMP protein family with 11 PPR motifs. The mutant allele crr4 (chlororespiratory reduction) exhibits reduced editing of the ndhD transcript (NAD(P)H dehydrogenase). It is speculated that CRR4 recognizes the target RNA and facilitates recruitment of general factors for RNA editing (C to U) events in the chloroplast [54]. A clear picture on RNA editing by the CRR4 protein has emerged by identifying its high-affinity binding to the neighboring region of the editing site of the ndhD-1 pre-edited transcript [82]. These findings have led to a working hypothesis according to which CRR4 functions as a trans-acting factor specifically interacting to a signature sequence near the ndhD-1 editing site and recruiting a putative editing enzyme such as cytidine deaminase probably via the C-terminal Eþ domain [82,93]. Except for the presence of the less-conserved C-terminal 15-aminoacid motif, CRR4 is similar in structure to CRR2, another PPR protein gene (At3g46790) that facilitates RNA splicing between rps7 and ndhB [42]. Because CRR2 belongs to the DYW group, it might contain all the domains of CRR4 and an additional DYW domain at the C-terminal end. Despite structural similarities, the two PPRs perform different RNA processing events. Whereas CRR4 is involved in RNA editing in chloroplasts, CRR2 probably recruits an endonuclease and performs RNA cleavage between the rps7 and ndhB cistrons [93]. All these studies strongly suggest multiple roles of PPR proteins in chloroplast RNA maturation processes, such as RNA trimming, stabilization, translation, and editing.

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7.1.2. PPR proteins in mitochondrial RNA processing Some nuclear-encoded RNAs are shared by cellular organelles such as chloroplasts and mitochondria for their gene expression processes [101]. Several nuclear proteins that regulate mitochondrial RNA metabolism belong to a growing family of proteins and the PPR protein family is one such important group. PPR proteins play possible roles in mitochondrial RNA metabolism in humans [45,60,75,76], yeast [69,70], fungi [19] and trypanosomes [77], but there is very little direct evidence in plants despite knowing that many PPR-containing proteins in Arabidopsis might be targeted to the mitochondria [6,66]. Although RNA editing takes place in both the mitochondria and chloroplasts, very few components of the machinery involved in plant mitochondrial transcript editing have been identified, compared with the chloroplast system because of the lack of a suitable mitochondria transformation method, reliable in vitro or in organello systems, and the presence of many editing sites. Plastid and mitochondrial editing processes share some common features; for example, the 20 nucleotides upstream of the editing site seem crucial in the C to U conversion, probably serving as the cis-sequence for binding of the trans-acting PPR protein. Recently, a genetic approach has identified two quantitative trait loci (QTLs) to be linked with differential RNA editing in Columbia (Col) and Landsberg eracta (Ler) ecotypes of Arabidopsis thaliana. These two QTLs were associated with two PPR genes At4g13650 and At4g14050, respectively, and might play an important role in editing of the mitochondrial transcript ccb206 C24 (orf206) [13]. Although there is very little direct evidence for the involvement of PPR proteins in editing-mediated mitochondrial transcript maturation, several PPR proteins in plants are associated with fertility restoration in CMS plants, a trait related to mitochondrial transcript processing (detailed in Section 7.2.2.). 7.2. PPR proteins in diverse functions Plant PPR proteins play an important role in organelle RNA metabolism, thereby regulating organellar gene expression and biogenesis [6,66,78,82,91e93,105]. Besides their importance in RNA processing, recent functional investigations on this group are revealing their diverse role in fertility restoration, embryo development, and plant development. With these diverse roles emerging, the importance of PPR proteins is being recognized in a much wider context. 7.2.1. PPR proteins and regulation of translation The translation process in plastids depends on nuclear and chloroplast interaction. About one-third of the ribosomal proteins required for translation in the chloroplast are encoded endogenously in the plastid genome whereas nuclear genes support the rest by either forming the translation apparatus or recruiting associated proteins to the translation machineries [10,23,109]. Nuclear-encoded PPR proteins aid either activation of the protein translation process [91] or recruitment of components of the translation engine in the organelles such as the chloroplast [105].

The maize PPR2 protein localized in the chloroplast stroma has been implicated in chloroplast biogenesis. Genetic and biochemical studies with the ppr2 mutant and protein show that the absence of PPR2 prevents accumulation of ribosomes in plastids. Compared with other plastome mutants of tobacco in which RNA polymerase activity was abolished, the effect of the ppr2 mutant was more severe. Because no RNA editing defects were detected, the possibility of the role of PPR2 in RNA editing was ruled out. The studies suggested that PPR2 functions in regulating translation by either processing plastidic ribosomal protein mRNAs or assembling the translation apparatus [107]. The role of PPR proteins as regulators of protein translation in the chloroplast was further substantiated in an in vivo experiment with the RIP-chip approach. The maize PPR protein CRP1 was directly associated with the 50 UTR region of both petA and psaC mRNAs [91]. Mutant crp1 showed reduced levels of both cytochrome b6f and PS1 core complexes [11], probably due to the defect in the translation of petA mRNA coding for cytochrome f and psaC mRNA coding for PS1. The close and specific interaction of the 50 UTR of these mRNAs and CRP1 strongly suggests that this PPR protein plays an essential role in regulating plastid mRNA translation by either influencing the local RNA structure near the start codon or recruiting a component of the basal translation machinery [91]. The RIP-chip technique also identified the maize PPR protein PPR4, which directly interacted with the rps12 transcript that codes for the ribosomal protein S12, a component of the 30S ribosomal subunit. PPR4 consists of a PPR domain with 16 tandem PPR motifs and an additional RNA recognition motif (RRM) domain at the N-terminal end. PPR4 is localized in the chloroplast stroma and functions as a trans-splicing factor involved in group II trans-splicing of the rps12 transcript, thus regulating translation in the chloroplast by ribosome biogenesis [92]. Although the exact role of the RRM motif in addition to the PPR motif has not emerged from the study, the RRM motif might be involved in RNA recognition and the PPR protein in recruiting splicing endonuclease. 7.2.2. PPR proteins restore fertility in CMS plants Cytoplasmic male sterility (CMS) is a maternally inherited phenotype characterized by the inability of a plant to produce functional pollen [84]. CMS has been observed in more than 150 plant species, including agriculturally important crops. Several sterilityefertility restoration systems have been employed to exploit heterosis [57]. The mitochondrial genes responsible for CMS are chimeric in structure [39,40]. Some of these genes encode a cytotoxic protein [105]. On the other hand, nuclear gene products restore fertility (Rf ) by altering the expression of CMS-associated genes in mitochondria. Thus, the CMS/Rf system in any plant is the result of the specific nucleoemitochondrial interaction of genes. Studies on the detailed mechanism of fertility restoration have identified some fertility restorer genes, namely, Rf2 of maize [20], Rf of petunia [12], Rfk1 (Rfo) of radish [14,25,52], Rf1 (PPR13) of sorghum [50] and Rf-1 [3,53,105] of rice. Except Rf2, the fertility restorer gene in maize, all other fertility restorer genes

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identified so far from different plants code for a PPR protein. The maize Rf2 encodes an aldehyde dehydrogenase protein [47]. The petunia Rf, radish Rfk1 (Rfo), and rice Rf-1 genes encode proteins consisting of 14, 16, and 18 tandem PPR repeats, respectively. All the proteins encoded by the four fertility restorer genes contain a putative mitochondrial targeting sequence in the N-terminal region. The putative mitchondrial targeting sequence of rice Rf-1 has the same set of seven amino acids in the N-terminal region as that in maize Rf-2, but there is no similarity between the target sequence of rice Rf-1 and sequences of Petunia Rf and radish Rfk1 (Rfo). Hence, the mitochondrial targeting sequence encoded by restorer genes is conserved, depending on the degree of evolutionary relationship of species rather than on the similarity of the restoration mechanism. The mechanisms of fertility restoration differ between PPR protein genes and other restorer protein genes. All fertility restorer genes encoding PPR-containing proteins reported so far alter the expression of CMS-associated genes [3,12,47,48,52]. In contrast to the PPR restorer, maize Rf2, which encodes aldehyde dehydrogenase, does not affect the accumulation of the CMS-associated protein URF13 [26] but instead compensates the metabolic scarcity [59]. The Petunia Rf, radish RfK1 (Rfo), and rice Rf-1 prevent the accumulation of the CMS-associated PCF (petunia fused gene) protein [80], ORF125 protein [52], and atp6 RNA [49], respectively. The rice fertility restorer gene Rf-1 of Boro II (BT) cytoplasm or ms-bo CMS is responsible for the processing of aberrant atp6 RNA of mitochondria when introduced into a CMS line [49]. A recent study with the rice fertility restorer Rf-1 locus provided a mechanistic detail about the fertility restoration process in the BT CMS system [106]. The CMS phenotype in rice with the BT cytoplasm was, because of a cytotoxic peptide, coded by the orf79 and B-atp6 genes. In the study, the classical Rf-1 locus was found to be composed of two genes Rf-1A and Rf-1B, both coding for PPR proteins with mitochondrial targeting sequences. Studies show that Rf-1A and Rf-1B restore fertility by two different mechanisms: the Rf-1A protein carries out nucleolytic splicing of the B-atp6/orf79 mRNA in three different regions, whereas the Rf-1B mediates silencing by complete degradation of the same mRNA. Rf-1A is epistatic over Rf-1B. In addition, Rf-1A might play a role in editing of the atp6 mRNA [106]. The radish fertility restorer gene Rfo is a nuclear gene that restores Ogura (ogu) CMS in Brassica napus [14]. It codes for a 687-amino-acid protein consisting of 16 PPR motifs and a predicted mitochondrial targeting pre-sequence. When introduced into the B. napus ogu cytoplasmic male sterile background, the Rfo gene restores male fertility. Similarly, the Kosena radish fertility restorer gene Rfk1 (orf687) encodes a 687-amino-acid protein having 16 PPR repeat motifs [52]. In male sterile plants the allelic gene of Rfk1 is altered by four amino acid substitutions in the second and third PPR repeats, stressing the importance of the domain formed by these PPR repeats in fertility restoration. Although the expression level of the CMS-associated mitochondrial transcript ORF125 remains unchanged, the protein is reduced significantly in fertility-restored plants. The product of Rfk1 might be responsible

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for lowering the ORF125 protein by binding directly or indirectly to the 50 region of the orf125 transcript and suppressing its translation [52]. A fertility restorer locus Rf1 has been identified in sorghum that encodes PPR13, a PPR protein with 14 repeat motifs. The Rf1 gene (PPR13) of sorghum may restore fertility in the A1 cytoplasm of sorghum. The PPR13 protein consists of a C-terminal E motif, which might play a role in post-transcriptional RNA processing [50]. 7.2.3. PPR proteins in embryogenesis Embryo-defective phenotypes of several mutant PPR genes imply their possible roles in plant embryogenesis [66]. In animals, the notable example of a PPR protein involved in embryo developmental process is the Drosophila Bicoid stabilization factor (BSF) protein with 7 PPR repeats. BSF is involved in stabilizing the bicoid mRNA, which is translated into the Bicoid protein, a morphogen required to establish the anterior-posterior body patterning during early embryogenesis [66]. In plants, many PPR proteins have essential and non-redundant roles in early embryogenesis-related processes. The PPR protein EMB175 identified from the Arabidopsis embryo-defective175 (At5g03800) mutant line plays an essential role in early embryo development [21]. In the mutant emb175, embryo development is arrested at a very early stage before the globular to heart transition. In addition to EMB175, six other PPR genes have been identified from T-DNA knockout mutant lines and have an essential and non-redundant function in embryogenesis. EMB175 is an 896-amino-acid PPR protein with 14 PPR motifs and is targeted to the chloroplast. Characterization of the EMB175 gene establishes its function in organelle biogenesis or essential biosynthetic or metabolic processes of an organelle, and its disturbance leads to a lethal embryonic arrest [21]. The recently identified Arabidopsis GLUTAMINE-RICH PROTEIN23 (GRP23) provides evidence for the involvement of a PPR protein in embryo development [27]. The loss-of-function phenotype of grp23 mutants produces embryonic growth arrests at the 16-celled dermatogen state, an early stage of embryo development. GRP23 is the only known PPR protein located in the nucleus, whereas the others are targeted to organelles. Functional analysis of GRP23 gene shows that it is expressed mainly in the embryo, endosperm, and gametophytes. Structure and domain analyses suggest that GRP23 acts as a transcriptional regulator. The GRP23 protein might interact physically with subunit III of RNA polymerase II through its C-terminal Gln-rich WQQ domain and also bind directly to cis-regulatory elements of DNA through its N-terminal basic domain [27]. Although the exact role of GRP23 in embryogenesis has not been elucidated from interaction studies on the WQQ domain with the RNA polymerase II subunit, GRP23 is speculated to play role as a transcriptional regulator. Another T-DNA insertional mutation in an Arabidopsis gene, At1g53330, coding for a PPR protein produced semilethal embryo-defective plants with arrested embryo development during the transition from the globular to heart stage [51]. Mutation in Arabidopsis (AtPPR4) (At5g04810), a homolog of the maize PPR4, causes embryo lethality, whereas

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maize ppr4 and ppr2 mutants show defective plastid ribosome machinery and translation, suggesting that the embryo lethality trait is a consequence of a defect in the plastid translation machinery [91]. The fact that most embryo-lethality-associated PPR proteins are targeted to the chloroplast supports the assumption. 7.2.4. PPR proteins regulated by circadian rhythm The circadian clock or biological clock is an inherent mechanism of an organism by which it maintains a regular function, in a cycle, with a periodicity of about 24 h. The biological clock of an organism can be synchronized in response to environmental stimuli. Several essential processes of life, including biochemical, physiological and behavioral processes, are governed by circadian rhythms. Higher plants exhibit many physiological processes such as leaf movement, stem elongation, stomata opening, photosynthesis, flowering, and gene transcription in circadian rhythms [31,36,41,73,89]. In Arabidopsis, several studies have firmly established the molecularegenetic basis of regulation of flowering time by the circadian clock [62,95,98]. The Pharbitis nil is an ideal plant system to study photoperiodic induction of flowering. The gene product of PnC401, a PPR protein, oscillated in a circadian rhythm [81]. A homologous gene of PnC401 identified in Arabidopsis encodes a PPR protein. A conserved domain of 12 PPR motifs (with 61% identity) is present in both the proteins and designated as the C401 domain. The only difference between the AtC401 and the PnC401 is the presence of the Nterminal serine threonine protein kinase domain in the former. The kinase domain might play an important role in phosphorylation of other proteins recruited by the PPR motif in AtC401 [81]. The protein structure of AtC401 is an excellent example of a combination of domains in which the known function of one domain (the kinase domain) might help in the functional annotation of an unknown domain (the PPR domain) [61]. Also, a 246-bp promoter fragment of the AtC401 gene could confer circadian rhythmic expression to a luciferase reporter gene, indicating that the AtC401 protein is regulated by a circadian rhythm [81]. 7.2.5. PPR proteins in organ development and boundary demarcation The aerial structure of higher plants is derived from the flanking cells in the shoot apical meristem (SAM). Throughout the life of a plant, the SAM produces stem tissues and lateral organs and regenerates itself. For correct growth, plants must maintain a constant flow of cells supplied by the meristem, the input of dividing pluripotent stem cells offsetting the output of differentiating cells. Several genes such as NO APICAL MERISTEM (NAM ), CUC1 (Cup-Shaped Cotyelodon1), CUC2, CUC3, CUP (Cupuliformis), and LOB (LATERAL ORGAN BOUNDARIES ) have been identified, which are responsible for partitioning of the lateral organ primordia from the SAM [1,2,94,99,102,106]. These genes interact in a complex manner to specify formation of lateral organ boundaries [2]. Most of these genes, except LOB, code for an NAC domain transcription factor. LOB constitutes a family of which it is the founder

member and codes for a LOB domain protein specific to plants [94]. We have identified a mutation in the gene LOJ (At2g39230) [84] from Arabidopsis T-DNA promoter trap lines [86]. The LOJ gene encodes for a protein with 19 PPR motifs preceded by a mitochondrial localizing signal sequence. In the loj mutant, a single T-DNA insertion at the At2g39230 locus exhibited GUS activity in the shoot apical meristem and all LOJs. Reporter gene expression patterns suggest that the LOJ gene is expressed in the boundary between the cotyledonary leaves of two-day-old seedlings and later in all LOJs of an adult Arabidopsis plant. Analysis of the loj mutant did not show any obvious morphological phenotype; however, the specific expression pattern of this gene and the promoterereporter fusion expression specificity suggests that LOJ might have a potential role in organ separation or in other aspects of lateral organ development [83]. One reason for not finding any mutant phenotype could be the presence of a functionally redundant gene, one (At3g54980) which exhibits approximately 59% amino acid sequence similarity and encodes for a PPR protein [88]. Further strategies such as generating a double mutant, gene silencing, and other functional analyses, may provide leads to the function of LOJ and other related genes. 8. Evolution of models for PPR functioning in plants Since their identification, PPR proteins have been predicted to be RNA-binding proteins because of their repeat structures producing a concave surface and facilitating binding of an extended hydrophilic and acidic ligand [66,96]. The proposition was extended further by several in vitro studies using recombinant E. coli and yeast to demonstrate that different PPR proteins are capable of binding to specific target RNAs [46,56,68,74,78]. Except for GRP23, which is located in the nucleus and probably binds DNA molecules [27], most other PPR proteins are targeted to cellular organelles and control organellar gene expression [6,37,56,66,82]. An in vitro study of RNA binding and UV crosslinking established that the petB transcript unit was a target for the HCF152 PPR protein [74]. Expression studies with the truncated HCF152 protein in prokaryotic systems revealed that PPR repeat motifs are crucial in determining the specificity of target RNA binding. At least six PPR repeat units forming a tunnel are required to fit a single-stranded RNA molecule [78]. Analysis and genetic characterization of plant PPR mutants are consistent in implicating PPR proteins in organellar RNA metabolism. RNA processing may involve all post-transcriptional processes such as mRNA splicing, maturation of 50 and 30 ends, RNA editing, and stabilization [6,11,35,42,54,74,82,92,108]. The evidence suggesting the involvement of PPR in almost all stages of organellar gene expression, and the fact that most PPR proteins lack any specific catalytic domain, have led to the proposal of an adapter hypothesis, according to which PPR proteins play the role of an adapter to recruit some additional factors to a specific site on the target RNA to regulate RNA processing [6,66]. A significant understanding of PPR proteins has revealed their role in molecular processes by the recent classification of PPR proteins into different subclasses and

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identification of motifs other than PPR motifs [61,87]. In general, the PCMP subfamily of PPR proteins are more variable because of their C-terminal domains, which might play an important role in recruiting other interacting factors such as endoribonuclease or cytidine deaminases [82,93]. Other functional domains present in PPR proteins (e.g., kinase or RRM domain) have a potential role in either activating or facilitating the assembly of factors for RNA processing events [81,92]. Apart from the processing of specific RNAs, evidence of the involvement of a PPR protein in the assembly and/or accumulation of ribosomes in plastids comes from studies on the PPR2 protein of maize [107]. Furthermore, an Arabidopsis PGR3 protein was speculated to be involved in both stability and in translation of petL mRNA through alternative sets of PPR repeat motifs [108]. The RNA-binding ability of the PPR protein was confirmed by both in vitro and in vivo studies using the RIPchip approach [91]. The results led to the proposal of a model in which a single-stranded RNA might bind between the conserved motifs of a monomeric or dimeric PPR protein surface. Moreover, the interaction of CRP1 is direct and specific with 50 UTRs of petA and psaC mRNAs, suggesting strongly the role of the CRP1 protein in the regulation of translation, possibly by influencing the local RNA structure or by assembling components of the basal translational apparatus [91]. In addition to their role in organellar gene expression and transcript processing, PPR proteins seem to be involved in embryogenesis and plant development too. Several T-DNA mutants in PPR genes exhibit impaired embryo development, may be because mitochondria and plastids are essential in providing energy to plant cells, particularly the actively dividing cells of the embryo [66]. However, the recent identification of EMB175, a PPR protein involved in embryo morphogenesis, provides a model of PPR protein function in a broader context [21]. Developmental and reverse genetic approaches have shown that EMB175 and six additional genes have a dramatic effect on embryogenesis. An alternative model states that a mutation in the PPR protein might have a direct impact on morphology, probably due to defective mRNA related to development, thereby disturbing some yet-undefined developmental process [27]. More recently, the identification and analysis of an Arabidopsis mutant grp23 provide strong evidence that a PPR protein is involved in early embryo formation [27]. Studies on the embryo lethal mutant caused by the T-DNA insertion in the AtPPR4 gene have pointed to the role of PPR proteins in the plastid translation process [92]. All these results indicate that PPR proteins play a very complex role in a variety of biochemical processes and are involved in diverse plant developmental processes; therefore, a simplistic model might not be sufficient to explain their mode of action. 9. Conclusions Various combinations of interacting RNAeprotein partners are the major contributors of development in eukaryotes and plants. This necessitates the thorough understanding of different RNA-binding proteins (RBPs) and their targets. PPR proteins are one such major group of plant RBPs and have

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a towering presence in plants, compared with other eukaryotes. Various studies on the functional role of PPR proteins have revealed their critical role in mitochondria and chloroplast biogenesis. Evidence indicates that PPR proteins are involved in all possible stages of RNA processing, including editing, maturation, stability, and translation in plant organelles. With more functional studies, the working model of PPR proteins is slowly emerging. PPR proteins appear to act as transacting factors, specifically recognizing a signature sequence in the RNA and facilitating recruitment of other factors for RNA processing and translation. Recent studies also highlight the role of PPR proteins in several diverse and important plant developmental processes such as fertility, embryogenesis, and organ separation; however, the mechanistic details of these processes are yet to be understood. Functional and genetic approaches with T-DNA knockout mutants and comparative genomics have already helped identify and characterize approximately 15 PPR proteins in plants. Approaches such as gene expression, cellular localization studies, antisense or RNAi suppression, and over-expression of PPR genes are some potential strategies being increasingly used to explore and study functions of PPR proteins in plants. Functional validation of these proteins, identification of the RNA and protein interacting factors, and unraveling of the detailed mechanisms may become more fruitful with routine applications of more advanced approaches, such as protein structure analysis, subcellular fractionation, immunoprecipitation, microarray, yeast hybrids, and other proteomic techniques. Another interesting consideration is to identify the presence of any ligand-specific domain in the RNA molecule that may facilitate interaction with PPR proteins. A comparative domain mapping of PPR proteins and understanding of various other motifs present in PPR proteins can give valuable information on the elucidation of PPR gene function. With increasing research in this field, in the coming years we may not only find an answer to the paradoxical question of why higher plants contain so many PPR genes in their genome but also understand how PPR proteins help regulate the organellar gene expression and plant development. Acknowledgements The authors gratefully acknowledge the financial assistance under NATP, CGP Grant (CGPII/253). DS and AMP gratefully acknowledge the financial support of ICAR and CSIR, respectively. We are grateful to Dr Vani Shankar for editing the manuscript. References [1] M. Aida, T. Ishida, H. Fukaki, H. Fujisawa, M. Tasaka, Genes involved in organ separation in Arabidopsis: An analysis of the cupshaped cotyledon mutant, Plant Cell 9 (1997) 841e857. [2] M. Aida, M. Tasaka, Morphogenesis and patterning at the organ boundaries in the higher plant shoot apex, Plant Mol. Biol. 60 (2006) 915e 928. [3] H. Akagi, A. Nakamura, Y. Yokozeki-Misono, A. Inagaki, H. Takahashi, K. Mori, T. Fujimura, Positional cloning of the rice

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