The RNA world of plant mitochondria

The RNA World of Plant Mitochondria MICHAELA H O F F M A N N JOSEF K U H N , KLAUS D A S C H N E R AND STEFAN B I N D E R

Molekulare Botanik Unive~sitiit Ulm 89069 Ulm, Germany I. The Origin of Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. A Monophyletic Origin of Mitochondria Is Indicated by Genomic Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Syntrophy-Based Symbiosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. ce-Proteobacteria Are the Closest Living Relatives of Mitochondria . . . D. Diversity of Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Transcription of Higher Plant Mitochondrial Genomes . . . . . . . . . . . . . . . . A. Mono- and Polycistronic Transcription Units in Higher Plant Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Identification and Characterization of Transcription Initiation Sites . . . C. Primary Structure Requirements for Plant Mitochondrial Promoters.. D. Protein Components of the Mitochondrial Transcription Machinery... E. Regulation of Transcription . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E Tissue-Specific Expression of Mitochondrially Encoded Genes . . . . . . . III. Processing of Plant Mitochondrial mRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . A. Complex Transcription Patterns Generated by 5 ~ and 31 Processing of mRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Intron Splicing in Mitochondrial Transcripts . . . . . . . . . . . . . . . . . . . . . . C. Messenger RNA Processing and Stability . . . . . . . . . . . . . . . . . . . . . . . . . D. RNA Editing by Base Modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Processing of tRNAs and rRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. A Variegated tRNA Population in Mitochondria of Higher Plants . . . . . B. Maturation of Mitochondrially Encoded tRNAs . . . . . . . . . . . . . . . . . . . V. Posttranscriptional RNA Processing Involved in Cytoplasmic Male Sterility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Mitochondria are well known as the cellular power factory. Much less is known about these organeUes as a genetic system. This is particularly true for mitoehondria of plants, which subsist with respect to attention by the scientific community in the shadow of the chloroplasts. Nevertheless the mitochondrial genetic system is essential for the function of mitochondria and thus for

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MICHAELAHOFFMANNET AL. the survival of the plant. In plant mitochondria the pathway from the genetic information encoded in the DNA to the functional protein leads through a very diverse RNA world. How the RNA is generated and what kinds of regulation and control mechanisms are operative in transcription are current topics in research. Furthermore, the modes of posttranscriptional alterations and their consequences for RNA stability and thus for gene expression in plant mitoehondria are currently objects of intensive investigations. In this article current results obtained in the examination of plant mitochondrial transcription, RNA processing, and RNA stability are illustrated. Recent developments in the characterization of promoter structure and the respective transcription apparatus as well as new aspects of RNA processing steps including mRNA 31 processing and stability, mRNA polyadenylation, RNA editing, and tRNA maturation are presented. We also consider new suggestions concerning the endosymbiont hypothesis and evolution of mitochondria. These novel considerations may yield important clues for the further analysis of the plant mitochondrial genetic system. Conversely, an increasing knowledge about the mechanisms and components of the organellar genetic system might reveal new aspects of the evolutionary history of mitochondria. © 2001 AcademicPress.

The mitoehondrion is one of three compartments of a plant cell carrying genetic information. The comparatively large and complex plant mitochondrial genomes (also called chondriomes) code for about 50-60 genes. Although only a tiny fraction of the estimated total 800-1000 mitochondrial proteins is encoded in the organellar DNA, mitochondrial genes are crucial for the function of these organelles and they therefore have to contain a complete genetic system for the expression and inheritance of their genetic information. In recent years much progress has been made in the elucidation of the land plant mitochondrial genetic information. The complete sequences of the mitochondrial DNAs (mtDNAs) of the Spermatophyta Arabidopsis thaliana and Beta vulgaris and of the liverwort Marchantia polymorpha have now made the entire coding capacities of three different plant species available (1-3). These data now form a solid basis for a comprehensive analysis of the expression of this genetic information and the underlying processes of regulation. Studies in various higher plant species show that transcription but especially the diversity of RNA processing plays a crucial role in gene expression and its regulation. The surprisingly great variety of distinct RNA processing steps reflects the complex and still-puzzling RNA world in plant mitochondria. To put the various forms and features of transcription and RNA processing in plant mitochondria into context we will begin from the historical perspective. History here implies the roots and evolution of the mitochondrial organelle. This view should help to profile and interconnect the diverse features in the plant mitochondrial RNA world.

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I. The Origin of Mitochondria According to the generally upheld endosymbiont theory, mitochondria and chloroplasts are descendents of free-living eubacteria-like organisms that have somehow become embedded into a host cell. During the development from the newly incorporated symbionts to the temporary organelles, the vast majority of the original genetic information was either lost or transferred to the host genome. Molecular and biochemical data convincinglyindicate that chloroplasts share a common ancestor with contemporary cyanobactefia, although the number of original endosymbiontic events is still a matter of discussion. Recently a comparative study of nuclear genes provided evidence for a single origin of the chloroplasts in red and green algae and, with limitations, also in glaucophytes (4, 5). Numerous subsequent secondary endosymbiontic events further distributed chloroplast DNA throughout many other enkaryotes (6).

A. A Monophyletic Origin of Mitochondria Is Indicated by Genomic Data An analogous singular origin of mitochondria is supported by several lines of physiologic, biochemical, structural, and genornic evidence. Structural and genomic analyses show a similar clustering of mitochondrial rRNA and proteincoding gene sequences, an overall comparable gene content, as well as similar derived gene arrangements observed in mitochondrial genomes of different lineages. Ribosomal protein gene orders, for example, are conserved in many mitochondrial genomes of algae and protists as well as in the liverwort Marchantia polymorpha and it is highly unlikely that such derived characteristics could be the result of convergent evolution (6, 7). Mitochondrial DNA-specific gene clusters are also found in the so far most ancestral mitochondrial genome in Reclinomonas americana (8, 9). The mitochondrial genome of this freshwater protozoan encodes 97 different genes, at present the by far largest number of genes detected in any mitochondrial genome. While 44 of the total 62 protein-coding genes have been found variously in one or more previously analyzed mtDNAs, 18 genes have not otherwise been observed. The observation that the Reclinomonas mtDNA is itself highly reduced compared to its hypothetical eubacteria-like progenitor and that it contains all of the protein-coding genes identified variously in other, more reduced mtDNAs substantiates the monophyletic origin of all mitochondria. Among the newly identified genes, those encoding eubacterial-like RNA polymerase subunits and secY homologs are especially noteworthy since they represent components of classical bacterial functions that have most likely become extinct in all other mitochondrial lineages investigated so far. The presence of a secY-like protein implies that Reclinomonas uses a protein sorting system similar to those

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in eubacteria and plastids. Such a secY-based import machinery has not been observed in mitochondria of any other organism. Particularly in the context of this article, the most intriguing feature of the Reclinomonas mtDNA is the presence of genes encoding all four components of a eubacteria-type RNA polymerase. Whereas such reading frames (with the exception of rpoD) are part of the standard set of plastid genes, only nuclear-encoded RNA polymerases have so far been described for mitochondria. Although it is at present unclear whether these eubacteria-type RNA polymerase genes in Reclinomonas are functional, they indicate that mitochondria once did contain an RNA polymerase that most likely derived directly from the 0t-proteobacterial endosymbiont but has been lost and replaced by single-subunit RNA polymerases in all other mitochondrial genomes investigated.

B. Syntrophy-Based Symbiosis While there is no question about the endosymbiont theory as such, several new hypotheses dealing with the initial organisms involved in the biogenesis of the eukaryotic cell and the driving force of this process have been put forward. Until recently it was thought that an aerobic bacterium was taken up by an anaerobic, so-called proto-eukaryotic cell and that the intimate relationship of (endo)symbiont and host was stabilized through mutual advantage, providing the latter with ATP and the engulfed bacterium and evolving endosymbiont with metabolizable substrates and physical protection (10-12). New developments have now emerged from the wealth of sequencing data that have become available in recent years. Sequences of nuclear genes encoding mitochondrial and hydrogenosomal proteins and the analysis of genes resembling the former in amitochondrial organisms indicate a relationship between mitochondria and hydrogenosomes and suggest also that even those eukaryotic organisms lacking these organelles once contained the respective precursor. This pushes the origin of the two organelles back to earliest eukaryotes and back to the birth of the eukaryotic cell. It also gives impetus to the hydrogen hypothesis favoring syntrophy as a driving force for the initiation of the endosymbiotic process and the origin of the eukaryotic cell per se (7, 11, 12). In this model an anaerobic ot-proteobacterium excreting hydrogen and carbon dioxide as waste products of anaerobic fermentation came into close contact with an autotrophic methanogenic archaean partner using hydrogen and carbon dioxide as sole sources for energy and carbon (13). The subsequent absence of hydrogen in the environment generated a selective force for a closer association between the two partners, now with the host fully depending on the waste products of the symbiontic bacterium, a development favoring an enlargement of the surface contact between the two partners. Gene transfer from the symbiont to the host genome then enabled the latter to provide substrates for the almost completely engulfed symbiont, which was now physically isolated from

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the surrounding environment. This process was accompanied by a change of the host from autotrophy to heterotrophy, a process which is criticized as a weak point of the hypothesis (11). When the host irreversibly became heterotrophie its metabolic dependence on hydrogen vanished, and so its dependence on anaerobic environments. Hydrogen again became a waste product and the utilization of oxygen in respiratory ATP synthesis could have become advantageous. This could have occurred in parallel with the increase of free oxygen in the atmosphere (13). The hydrogen hypothesis shares some features with another syntrophy-based hypothesis, which also favors a methanogenic aiehaeon as original host and an anaerobic bacterium as symbiont and as antecedent of mitochondria (14, 15). But unlike the hydrogen hypothesis, here a sulfate-reducing &proteobaeterium is considered as the primary symbiont which was simultaneously or shortly later joined by an o~-proteobaeterial methanotroph as an additional second symbiont. Although many aspects of these new hypotheses sound very reasonable, there are intensive discussions among evolutionary biologists and many of them still seem to favor the original hypothesis by Margulis (6, 11).

C. 0~-ProteobacteriaAre the Closest Living Relatives of Mitochondria Just as the single origin of mitochondria is generally accepted, there is little dispute that extant oe-proteobaeteria are the closest relatives of these organelles. This is impressively confirmed by several features in the genome of Rickettsia prowazekii. Extensive similarity observed in the comparison of amino acid sequences of nuclear-encoded mitoehondrial proteins of yeast with those encoded by the Rickettsia bacterial genes intriguingly showed the close relationship of this contemporary intraeellular bacterium with mitochondria. In addition the functions of the proteins encoded by Rickettsia prowazekii and mitochondria are strikingly similar, both lacking genes for the anaerobic metabolism of sugars (16, 17). Another common feature of the Rickettsia and the mitoehondrial genomes is the high amount of noncoding sequences. While in the parasitic bacterium most of these noncoding sequences are probably inactivated genes on their way to extinction, the mitoehondrial sequences without any obvious function, which account for about 60% in both Arabidopsis and sugarbeet, are at least partially the result of sequence duplication, integration of retrotransposon, and/or DNA from the plastids and the nucleus. The origins of the large amounts of noncoding sequences in mitochondria and Rickettsia thus appear to be different. Despite the compelling similarities a direct origin of mitoehondria from Rickettsia is highly unlikely and other comparisons of mitoehondrial proteins identify Rhodospirillum and Bradyrhizobium, both ot-proteobacteria, as next extant relatives of mitochondria (6).

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D. Diversity of Mitochondria What are the consequences of the above reflections on the origin of mitochondria, particularly in the framework of this article? The eubaeterial origin of mitochondria anticipates that at least some components of the mitoehondrial genetic system and the respective tlNA world shows eubacterial characteristics. However, since for present-day mitoehondria almost all components of the genetic system are nuclear encoded, they may have originated from the endosymbiont or they may have alternatively been recruited from the arehaean or a proto-eukaryotic cell and become adapted to the symbionts requirements. This could be the case of the 3' processing of mitochondrial tRNA precursors, where a single cut occurs exactly at the 3' end of the pre-mature tBNA. Such a mode of tRNA 3' processing is employed in the nucleus but is not observed in eubacteria, where, for example, in Escherichia coli, a series of endoand exonucleolytie activities is involved in this ttlNA maturation step. It would be interesting to know how in comparison this processing step is performed in arehaea. There are also examples, however, where an assignment to one of the potential progenitors is not possible as, for example, in the instance of the nuclear-encoded mitochondrial RNA polymerases of the phage type. In general, and as intriguingly demonstrated by the BNA polymerase conundrum, clear characteristics of one or the other contributor of genetic information to the eukaroytic cell are often not obvious or simply do not exist. This is due to the tremendous diversity of mitochondria and their DNAs. The extremes of mitochondrial genomes range from the more ancestral types with the by far most primitive so-called proto-mitoehondrial genome of Ileclinomonas and other protists as well as of land plants, to the highly derived types, for example, present in animals, fungi, some green algae, and Plasmodium (7). A comparatively wide variation also exists in the modes of expression mechanisms. Beside some parallels, for example, the single-subunit-type BNA polymerase or a mitoehondrial RNA heliease, homologs of which have been described in animals, yeasts, and plants, there are plenty of different modes of t/NA processing, each offering the possibility for regulation of gene expression and contributing to the fascinating BNA world in mitoehondria.

II. Transcription of Higher Plant Mitochondrial Genomes A. Mono- and PolycistronicTranscription Units in Higher Plant Mitochondria The about 50-60 genes generally found in higher plant mitoehondria have been identified in various plant species and their transcription has been studied

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by Northern blot, primer extension, and S1 nuelease protection analyses. These studies revealed both mono- and polyeistronie transcription units, some of which are conserved among a number of plant species. For example, the rp15-rps14cob tricistronic operon has been identified in pea, rape seed, potato and Arabidopsis thaliana, with the latter two species having retained only pseudogenes of rps14 downstream of the rpl5 reading frame (18-22). These gene arrangements have, on the other hand, been partially or completely dismantled in Oenothera and Vicia faba, where recombination has dissolved the conserved gene arrangements and created new gene orders and species-specific transcription units (23-25). The complete sequence of the mitoehondrialgenome ofArabidopsis thaliana allowed a comprehensive exemplary survey of gene distribution within a plant mitochondrial genome (1). Determining gene order in the mitoehondrial genome of a given plant species is faced with the problem of genome rearrangements by homologous recombination within the DNA. The Arabidopsis chondriome contains one set of direct and inverted repeats each, which are predicted to recombine into five different configurations. Four of them were confirmed by sequencing of the connections into the various flanking regions of the repeats and were found to be present in equimolar amounts (26). Such recombinations lead to partially differing linear gene orders in the different configurations. The gene arrangement of one of three configurations representing the entire genomie sequence allows meaningful conclusions about potential mitoehondrial transcription units. The 58 genes encoded in the 366,924 bp of the genome define intervening spacer sequences of highly variable lengths. While the theoretical mean distance between two genes is calculated to be about 6.5 kb, noneoding sequences of up to 57 kb are actually observed. About 40 of the genes encoded are part of clusters with two or more reading frames, where internal spacer sequences are 3 kb or less. Although these distances per se do not allow a definite conclusion about eotranscription, the genes within these clusters and thus the majority of the genes in the mitochondrial genome of Arabidopsis thaliana are most likely transcribed as part of multipartite transcription units (27). A similar clustering of genes is observed in the sugar beet mitochondrial genome, although the gene order differs completely from the Arabidopsis genome (2). Multieistronic transcription units thus seem to be the rule rather than the exception in higher plant mitoehondrial genomes, although the genome is so large. A detailed complete transcription map of one of the completely sequenced mitochondrial genomes is now necessary to exactly define all of the transcription units in a higher plant mitoehondrial genome. Multicistronie transcription units are typical for prokaryotes and thus may reflect the prokaryotic origin of mitochondria, even though the actual mitochondrial transcription units are completely different from those in contemporary bacteria and furthermore vary greatly among different plant species.

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Monocistronic transcription units, which are characteristic for eukaryotes, are also found in plant mitochondria but may have been secondarily generated with the now solitary genes having become isolated during evolution of the mitochondrial genomes through extensive recombination as well as by the integration of promiscuous DNA of chloroplast and nuclear origin (1).

B. Identification and Characterization of Transcription Initiation Sites As a consequence of the frequent multicistronic transcription units, mitochondrial genes are often transcribed into very complex mRNA patterns. Surprisingly, solitary genes with no obvious transcription partner also are represented in multiple RNAs. This plentitude of transcripts with numerous different 5' ends has considerably complicated the identification and characterization of mitochondrial promoters. To circumvent these difficulties, and to select promoters unambiguously, in vitro capping analyses have been employed for several plant species (28-32). Taking advantage of the selectivity of the guanylyltransferase, the capping enzyme, and the observation that none of the mitochondrial transcripts are capped in vivo, this technique allows the selective labeling of primary transcripts, which derive from de novo transcription initiation events and which therefore carry triphosphate groups at their 5' ends. This labeling procedure has made the primary transcripts amenable for selective investigation and allowed the unambiguous identification of several transcription initiation sites. These analyses revealed that multiple promoters as well as various processing events contribute to the steady-state complexity of plant mitochondrial transcription. Extreme examples, for instance, are found in maize, where such investigations identified three and six separate transcription initiation sites for the cox3 and atp9 genes, respectively (28, 29). Likewise, several promoters are found upstream of the cox2 gene in the same plant, where this gene can be either connected with an upstream sequence containing three promoters or can have recombined with a 5' region containing two other transcription initiation sites, one of which is composed of seven overlapping promoter units (33). Visual comparison of the sequences surrounding these transcription initiation sites revealed loosely conserved sequence features, which were consequently suggested to be at least part of the plant mitochondrial promoter identity elements. A 5'-CRTA-3' tetranucleotide seems to be part of almost all higher plant mitochondrial promoters. This motif is extended to the conserved nonanucleotide element 5'-CRTAAGAGA-3', which characterizes most of the initiation sites in dicot plant species. The identification of these motifs provided the basis for the identification and detailed characterization of plant mitochonclrial promoters by in vitro transcription systems (see below). While the situation at the 5~ends is thus becoming at least partially clear, virtually nothing is known about the termination of transcription. S1 nuclease

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protection experiments have identified several transcript termini immediately downstream of inverted repeats, and it was consequently speculated that these might have a function as terminators of transcription (34). Functional investigation of the double inverted repeat downstream of the pea atp9 gene in an in vitro transcription system, however, demonstrated that mitoehondrial transcription proceeds through this structure without impediments and gave no indication for transcription termination (35, 36). A subsequent investigation of several other plant mitoehondrial stem loop structures further substantiated that these structures are not connected to termination of transcription (J. Kuhn and S. Binder, unpublished results). In addition, functional analysis of the pea atp9 inverted repeat suggested that these structures were processing signals and thus explained their conserved structure and location at the termini of steady-state transcripts (36). Insufficient data are available on the origin of other mitochondrial 8' transcript termini to allow any conclusion about conserved primary or secondary structures that could be related to transcription termination and it remains open how transcription is terminated in this compartment of the plant cell. The functional assignment of the inverted repeats indicates that secondary structures resembling rho-independent transcription terminators of eubaeteria are not conserved in plant mitoehondria.

C. Primary Structure Requirements for Plant Mitochondrial Promoters The functional characterization of plant mitochondrial promoters has greatly benefited from the establishment of in vitro transeription systems in three different plant species. Initially established in wheat and subsequently also in maize and pea, the in vitro analysis of mutated DNA templates allowed the precise definition of primary sequence requirements for functional mitochondrial promoters (37-39). Analysis of the maize atpl promoter, for example, revealed that mutations of nucleotides between positions - 1 2 and +5 relative to the transcription start site generally decrease the rate of transcription initiation at this promoter (40). Within this region a central domain ranging from nueleotide positions - 7 to +5, encompassing the conserved 5'-CRTA-3' motif, and an upstream domain centered around positions - 1 1 / - 1 2 contain the nucleotides where the most severe effects on transcription initiation activity are observed upon mutation. An analogous analysis of two cox3 promoters in the same plant species showed that a 26-bp-long fragment contains full promoter activity, while a 14-bp element does drive correct transcription, but with only reduced activity. The significance of the central domain was confirmed in the cox3 initiation sites, but manipulations within the upstream element had only little or no effect on the transcription rate in vitro (41). Thus the structure of mitochondrial promoters seems to be only loosely conserved even within a single plant species,

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illustrating the variable character of mitochondrial promoters at least in this monoeot plant. More detailed examinations of other promoters may help to build a clearer picture of promoter requirements in this group of plants. In dicot plant species, the pea atp9 promoter was exemplarily investigated in a homologous in vitro transcription system (39). Detailed deletion studies upstream and downstream of the transcription initiation site identified an 18-bp element to be both necessary and sufficient for transcription in vitro (42) (Fig. 1). This sequence, which showed full promoter activity if cloned into alien sequence contexts, contains the highly conserved nonanucleotide motif 5'-CRTAAGAGA-3' ranging from positions - 7 to -4-2, in which transcription starts with a canonically conserved GA dinucleotide. An additional well-conserved motif is located between positions - 9 and -14. This element is characterized by its high content of adenosines and thymidines and has accordingly been termed the AT-box. Conversion of these nucleotides into their complementary sequence indicated that it is not the content of adenosines and thymidines per se, but rather the nucleotide identities at certain positions that are important determinants of promoter function (42). The two motifs, however, do not have any separable competence, since insertion of varying numbers of nucleotides between the nonanueleotide motif and the AT-box completely abolishes promoter function and thus shows that the two elements function in concert as a single entity in this promoter. A comparison of the - 1 4 to +4 sequences in 11 bona fide dieot promoters from various plant species identified five positions with 100% sequence conservation and eight positions conserved in 73% or more of the promoters compared. This compilation supplied the basis to design point mutations, which in functional in vitro tests basically confirmed the highly conserved positions to be the most important nucleotide identities for promoter function. The conversion, for example, of the 100% conserved guanosine to adenosine at position +1 reduced the transcription rate to less than 30% of the wild-type activity, thus confirming the importance of this nucleotide identity for promoter activity (M. Hoffmann and S. Binder, unpublished results). The compilation of the various promoters furthermore illustrates the considerable conservation of mitoehondrial promoters among different dieot species. This observation implies that the transcription machineries should be very similar in dieot plants, which was indirectly confirmed by correct transcription of heterologous DNA templates from soybean, Oenothera, and potato in the pea mitochondrial in vitro transcription system (39, and M. Hoffmann and S. Binder, unpublished results). The high conservation of this type of promoter in dieot plant mitoehondria was subsequently deployed to inspect the complete mitoehondrial sequence of Arabidopsis thaliana. This search identified 29 potential promoters, of which i6 are located within 3 kb or less upstream of identified genes (27). Besides

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the Arabidopsis 18S rRNA promoter, which was identified in a previous capping analysis, the detection of 5' transcript termini at two other potential transcription initiation sites supports their function as active promoters (43, and M. Zink, M. Hoffmann and S. Binder, unpublished results). As mentioned above, conserved promoter structures are also present upstream of 18S rRNA genes in some plants. In vitro transcription analyses of these suspected 18S rRNA promoters from several dicot plant species, however, yielded inconclusive results. While clear and reproducible signals demonstrate efficient transcription initiation at the potato 18S rRNA promoter, only weak and variable results were obtained with Oenothera, Arabidopsis, soybean, and the homologous 18S rDNA template in the pea in vitro transcription system. The reasons for these ambiguities are unclear and further in vitro studies are necessary to clarify the peculiarity of the 18S rRNA promoters. In maize, transcription initiation has likewise not been detected at the 18S rRNA promoter, suggesting different and/or additional parameters for promoter activity (44). Besides the group of well-conserved promoters with the distinctive high conservation in dicots, several cappable 5' transcript termini have been identified, where the surrounding sequences show no similarity to the conserved class of promoters and have no discernible similarity to each other. Such nonconserved transcription initiation sites are found, for example, directly at the 5' end of the mature 26S rRNA in potato, upstream of the soybean RNAe, and upstream of several tRNA genes in potato (31, 45-47). These non-consensus-type promoters are, however, not typical for tRNA genes since both in vitro capping and in vitro transcription analyses identified and verified a nonanucleotide-type promoter, for example, upstream of the clustered genes for tRNA Phe and tRNA Pr° in Oenothera (48). Several potential promoters of this conserved nonanucleotide type have also been found upstream of some of the tRNA genes in the Arabidopsis mitochondrial genome (M. Hoffmann and S. Binder, unpublished results). The existence of alternative promoters is also indicated by the lack of conserved promoter sequences in reasonable distances upstream of several genes and gene clusters in the Arabidopsis mitochondrial genome (27). An alternative promoter has also been described in the teosinte Zea perennis. This so-called conditional promoter is only active in the presence of the dominant Mct allele and shows only limited similarity to the consensus-type promoters of maize (49). Taken together these results suggest the presence of additional alternative promoter structures that might be served by an alternative transcription machinery. In contrast to chloroplasts, where eubacterial-type promoters have been described, the "prokaryotic" origin of mitochondria is not reflected by the promoter structure. Here promoter requirements have been adapted to another type of RNA polymerase, whose origin is still enigmatic.

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D. ProteinComponentsof the Mitochondrial Transcription Machinery Based upon the identification and the detailed characterization of several mitochondrial promoters (i.e., the cis elements), considerable progress has been accomplished in the elucidation of protein components (i.e., trans-acting factors) of the plant mitochondrial transcription machinery. Initiated by the availability of an expressed sequence tag (EST) from rice with significant similarity to the yeast mitochondrial RNA polymerase, partial sequences of phage-type RNA polymerases have been sequenced from various species (50). These comprise a broad spectrum of multi- and unicellular organisms including higher plants. The complete sequence of such a putative mitochondrial RNA polymerase was first determined in Chenopodium album (51). In Arabidopsis thaliana three distinct phage-type single-subunit RNA polymerases have been identified (52, 53). In organello and in vivo studies with respective GFP fusion proteins revealed that beside mitochondria, also chloroplasts are the target of this type of RNA polymerase (54). While two genes encode enzymes that are exclusively destined to mitochondria or chloroplasts, respectively, the gene product of a third gene seems to be targeted to both organelles (53). Mitochondria of dicot plant species thus contain at least two distinct RNA polymerases, while chloroplasts accommodate at least three, two nuclearly encoded single-subunit and a third plastid-encoded multisubunit eubacterial-type RNA polymerase. Recentlyyet another, fourth RNA polymerase activity has been suggested for chloroplasts, which further increases the complexity of the transcription machinery in this organelle (55). Three phage-type RNA polymerases, partial sequences of which have also been described in tobacco, seem to be unique for dicot plant species, since in wheat and maize, only two distinct mitochondrial and chloroplast proteins have been identified (56-59). A eubacteriallike RNA polymerase in mitochondria, which would be expected from the endosymbiont theory, has been discovered in the mitochondrial genome in Reclinomonas americana, but has so far not been found in higher plants. Further details of organellar RNA polymerases of higher plants have been summarized elsewhere and are thus not discussed in this review (60). Compared to the RNA polymerases much less is known about accessory factors of transcription in plant mitochondria. Considering the similarity among yeast, animal, and plant mitochondrial RNA polymerases and the similar compositions of the transcriptional machineries in the former groups of organisms, which includes at least one common essential transcription factor, it is highly plausible that analogous and even homologous proteins also exist in mitochondria of plants (61, 62). Moreover, the complexity of plant mitochondrial promoters makes it likely that more than one transcription factor will be required for transcription of all plant initochondrial genes.

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In pea and wheat, biochemical approaches have been employed to purify plant mitochondrial transcription factors. Using a denaturation-renaturation protocol including several purification steps such as hydroxylapatite, phosphocellulose, and reversed-phase high-pressure liquid chromatography, (at least) two polypeptides with apparent molecular masses of 32 and 43 kDa were isolated from pea mitochondria (63). Proteins with the same sizes were also purified by affinity chromatography with DNA fragments representing the pea atp9 promoter from the same plant species (C. Thalheim and S. Binder, unpublished results). While no definite N-terminal sequence could be determined from the 32-kDa protein, the larger polypeptide was identified as isovalerylCoA-dehydrogenase, a member of the acyl-CoA-dehydrogenase family (64). This protein is unambiguously located in the mitochondrial matrix and catalyzes the third step in leucine degradation in mitochondria (64a). Its participation in plant mitochondrial transcription, however, remains to be substantiated. Several examples of dual functions have been described for other proteins and an analogous parallel involvement in both biochemical pathways as well as mitoehondrial transcription initiation is possible for this 43-kDa polypeptide (65). In wheat a mitochondrial DNA-binding protein with a completely different apparent molecular mass (63 kDa) has been identified. This polypeptide was purified from transcriptionally active mitochondrial protein fractions, and peptide sequencing allowed the identification of a respective cDNA. The overexpressed protein exhibits DNA-binding activity with affinity to the cox2 promoter as well as upstream regions and indeed stimulates transcription initiation at this promoter in vitro. These features and its elevated expression in early developmental stages of the plant parallel to the cox2 transcription strongly suggest a functional role of this protein in transcription initiation in wheat mitochondria. A search ~br similar proteins in public domain databases identified several hypothetical mitochondrial proteins in Arabidopsis thaliana, confirming that such proteins are present also in this dicot plant, suggesting several proteins with similar functions in a single plant species (66). In line with the absence of bacterial multisubunit RNA polymerases in plant mitoehondria, there are no clear indications for eubacterial Sigma-like transcription factors in plant mitochondria. To elaborate the complete composition of the plant mitochondrial transcription apparatus, additional, most likely hard-core biochemical efforts might be necessary. Our present knowledge about mitochondrial proteins potentially involved in transcription is summarized in Fig. 1.

E. Regulation of Transcription Northern analysis of mitochondrial genes in various plant species showed that the steady-state levels differ considerably among individual RNAs. The steady-state amount of distinct RNAs can in mitoehondria as in all other genetic

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NNNNN[ -8 -7

-14 AT-Box

+2 CNM

+4 Pu

FIG. 1. Promoter structure in dicot plant mitoehondria and potential components of the plant mitochondrial transcription apparatus. In vitro transcription studies of the pea atp9 promoter region identified a sequence block extending from position - 14 to +4 that is both required and sufficient for autonomous promoter function independent of surrounding sequences. This sequence comprises the transcription start point (+ 1) indicated by a bent arrow. Although this promoter functions only as a single entity, three different prominent parts became evident: The highly conserved nonanucleotide motif (-7 to +2, CNM), the AT-box(-14 to -8, comprising at least two very important nucleotide identities at positions -12 and -11), and two nucleotides at positions +3 and +4 (Pu), of which at least one has to be a purine for full promoter function. Besides two RNA polymerases of about 110 kDa identified in Arabidopsisthaliana, mitochondrial DNA-binding proteins of 63, 43, and 32 kDa have been purified from mitochondria of wheat and pea. Homologous proteins of the wheat 63-kDa proteins are also present in Arabidopsisthaliana, suggesting an identical or similar function iu dicot plant species. However, their function as transcription factor(s) (TF) remains to be determined. One of the proteins binding to the pea atp9 promoter has been identified and characterized as isovalerylCoA dehydrogenase (IVD), which catalyzes the third step in the leucine degradation pathway. The immediate relationship of this protein to mitochondrial transcription initiation is unclear. systems be controlled by the transcriptional rate (i.e., RNA synthesis) and/or at the level of transcript stability, which is in turn influenced by posttranscriptional processes. These either increase the life span of an RNA or stimulate its degradation. To address the question of whether individual genes are transcribed at different rates in plant mitochondria, in organello RNA synthesis (run-on transcription) experiments have been employed. Most of these analyses were done with initochondria isolated from maize. In one of the first reports Finnegan and Brown (67) found that rRNA genes are transcribed at rates 5- to 10-fold higher than three protein genes tested for comparison, atpl, atp6, and cox2. Surprisingly, they also found that large noncoding sequence stretches with sizes up to 21 kb are actively transcribed, although no stable transcripts could be detected for these regions. Mulligan et al. (68) also identified the rRNA genes as the most strongly transcribed genes, with transcription rates about 2- to 14-fold higher than those for the atpl reading frame, which is the strongest transcribed protein coding gene, and for cox3, the gene with lowest transcriptional activity, respectively. The relative rates of RNA synthesis did not reflect the relative steady-state abundances of the RNAs, indicating that stability is an important factor influencing the steady-state level of a given RNA molecule. In addition the determination of the relative gene copy n u m b e r excluded this p a r a m e t e r as the sole source for the different transcription rates.

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Thus it was concluded that promoter strength may be an important constituent of transcriptional rate. In another report, the gene encoding ribosomal protein S 12 (rps2) was identified as the most strongly transcribed gene in maize mitochondria. The relative transcriptional rates of rrn26, rrnl8, atp6, and atpl differ significantly from the previous reports, indicating the difficulties in clear quantification of such in organeUo experiments (69). However, the tendency that rRNA genes are among the most strongly transcribed mitoehondrial genes in maize is basically confirmed. In this investigation no tissue specific differences were detected, but significant variation of apparent promoter activity was found at least for the rps2 gene, which is transcribed at a fivefold reduced rate in the Texas male cytoplasmic line B37(T). Comparison of the gene copy numbers in mitochondria of maize and Brassica hirta revealed a rough correlation with the transcriptional rates different from previous investigations (68, 70). A recent comprehensive run-on transcription analysis of all mitochondrially encoded genes from Arabidopsis thaliana also revealed significant differences of transcriptional rates among individual reading frames, even between genes that code for subunits of the same multiprotein complexes (71). These substantial differences are at least partially counterbalanced in the steady-state RNA by most likely posttranscriptional processing and different RNA stabilities. In contrast to maize, the rRNA genes in Arabidopsis thaliana are transcribed at levels comparable to those of protein-coding genes. This suggests that their quantitative dominance in the steady-state RNA is predominantly caused by their extraordinary stability. Taken together, all in organello experiments confirm distinct transcriptional rates for individual genes in plant mitochondria, although minor differences are observed among individual experiments. It also seems clear that the different rates originate from differences in promoter strength on top of the influence of the gene copy number. The exact parameters determining the strength of a promoter in vivo are unclear. An investigation of the maize cox2 promoters suggested that the long-range genomic context influences the activity of such promoters. In this instance identical promoters are present in two different genomic environments (A and B) 5' of a direct repeat which is either located upstream of cox2 or upstream of an apparent noncoding region. Southern blot and RT-PCtt analyses revealed that although the proportion of the genomes carrying regions A and B is 1:6, region A was used at a disproportionally higher rate in combination with the cox2 gene. It was therefore concluded that the activity of a promoter is influenced by its genomic context and that intergenomic recombination may regulate gene expression in plant mitochondria (72). Detailed in vitro mutagenesis studies of mitochondrial promoters revealed that alterations of individual nucleotide identities can greatly influence the transcriptional activity of a promoter, confirming that the primary structure is also

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a major determinant of promoter strength (40, and M. Hoffmann and S. Binder, unpublished results). The numerous variants found for promoter sequences in different genes and in different plants thus imply as a consequence different transcription rates. A clear in vivo correlation of promoter sequence variance to rate of transcription is, however, hampered by the above detailed presence of numerous promoters for a given gene and still needs to be clone thoroughly in at least one plant species. The relevance of such in vitro observations for in vivo promoter strength is unclear. Generally the run-on transcription experiments reveal and confirm that different transcriptional rates exist, but also that posttranscriptional processes to a large extent determine the steady-state levels of several RNAs and are thus a major control level of mitochondrial gene expression.

F. Tissue-Specific Expression of Mitochondrially Encoded Genes Tissue-specific gene expression is a conditio sine qua non for the development of all organisms and thus is a common phenomenon among nuclearencoded genes in all multicellular organisms. In the nucleus this is most often determined on the level of transcription initiation. The detailed investigation of three nuclear-encoded genes for subunits of the NADH dehydrogenase (i.e., 22kDa PSST protein, 28-kDa TYKY protein, and 55-kDa NADH-binding protein) in plants revealed strong transcription throughout flower development with a locally enhanced expression in anthers and pollen (73-75). This complex of the respiratory chain is composed of nuclear and mitochondrially encoded subunits and requires generally equal stoichiometric presence of the about 30-40 different proteins in the complex. Accordingly, an enhanced expression of nuclear genes should be accompanied by an increased expression of respective genes in the mitochondria. This could be accomplished by increasing the copy number of mitochondrial genes or by tissue-specific enhancement of mitochondrial gene expression. Only very few reports address these important topics. In one such analysis the copy number of the cob, cox2, and atpl genes per cell and their expression was quantitatively investigated in successive sections of wheat leaves (76). It was found that the abundance of these genes is 5- to 10-fold higher in the most basal section of the leaf in comparison to all other sections, which contain similar amounts up to the tip. In contrast, the relative abundance of mitoehondrial transcripts (cox1, cox2, cob, and atpl) successively decreases from the basal to more distal sections. A distinct discrepancy was observed for the atpl gene, the gene copy number of which decreases 5- to 10-fold, while its transcript levels decrease about 2.5- to 3.5-fold. These results indicate that enhanced copy number might accomplish an increased tissue-specific expression of mitochondrial-encoded genes, while

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the different levels of gene copy numbers and transcripts also indicate that other factors such as transcriptional rates or RNA stability are important for final steady-state levels of mitochondrial transcripts (76). A detailed analysis of cell-specific mitochondrial gene expression was also done in maize seedlings. Here the expression of atp6, 26S rRNA, and the autonomously replicating mitoehondrial RNA plasmid RNAb as well as the cytoplasmic 17S rRNA were compared by in situ hybridization and quantitative RNA dot blot hybridization during vegetative growth (77). These experiments revealed differences in the levels of mitoehondrial RNAs among different tissues and different cell types within the same organ of maize seedlings. For example, atp6 and RNAb are about twofold stronger expressed in leaves than in eoleoptiles. In contrast the 26S rRNA is found at twofold higher amounts in coleoptiles. This shows that different mitochondrial transcripts are expressed at different levels and that these differing levels cannot simply be explained by altered abundances of mitochondria or gene copy numbers. Altered transcriptional rates and/or stabilities must also be responsible for the tissue- and cell type-specific levels of transcripts. Tissue-specific alteration of transcription is also indicated by the observation that all mitoehondrial mRNAs are more abundant in vascular tissues than in surrounding parenchyma (77). Analogous observations have been made in other organs and tissues, from which increased mitochondrial transcript levels could be generally correlated with increased activity of cell division. In summary these investigations show that during vegetative growth, transcript levels of individual genes are regulated in a cell-specifical manner by a combination of various control mechanisms. Cell-specific expression connected to cytoplasmic male sterility (CMS) will be discussed in the respective section below.

III. Processingof Plant Mitochondrial mRNAs A. Complex Transcription PatternsGenerated by 5' and 3' Processingof mRNAs As stated above, numerous overlapping transcripts are observed for a large number of mitoehondrial genes. These include genes that are part of polycistronie arrangements or that contain one or several introns, but multiple transcripts are also found for singular reading frames. In this multitude of genes we will take a look at the atpl and atp9 loci in pea, which are located a short distance from each other but on opposite strands. The former, atpl, is transcribed into four different RNAs with differing 5' and 3' ends. This complexity may be at least partially due to a 1.7-kb repeated sequence that partially covers the atpl reading frame. The atp9 gene is transcribed into three different transcripts of

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? DNA

?

r-

5'

Transcription 1.3kbmRNA ¢

? 0.6 kb mRNA

0.4 kb mRNA

A

~ ~~/~

FIG. 2. Transcription pattern of the pea atp9 gene. This gene is transcribed into three RNAs with lengths of 0.4, 0.6, and 1.3 kb, respectively. All these transcripts have identical 3 r ends that have been mapped immediately downstream and within a double-stem-loop structure, which is encoded by a double inverted repeat (indicated by gray arrows in the linear genomic representation in the top line). The atp9 mRNAs have different 5 ~ends, with the most upstream terminus derived from de novo initiation at a conserved mitochondrial promoter (open boxes, transcription initiation is indicated by bent arrows). The origin of the 5~termini of the shorter transcripts is unclear. Besides additional transcription initiation events, they could also derive from endonucleolytic processing reactions (black triangles) or even from exonucleolytic digestion by an 5r-to-3 ' exoribonuclease (indicated by an open mouth).

1.35, 0.6, and 0.4 kb, respectively. While these transcripts differ in their 5' ends, they terminate at identical 3' ends, which map immediately downstream of an inverted repeat (35) (Fig. 2). In vitro transcription analyses revealed the 5' end of the largest atp9 RNA to derive from bona fide transcription initiation (39). No transcription initiation could be obtained, however, with a DNA template covering the region surrounding the 5' end of the shortest transcript, suggesting that this terminus derives from a processing event. This example highlights the difficulties encountered in defining the exact origin of a 5' end. The assignment of a transcript terminus as deriving from processing events is often made as an indirect argument in which the given end is simply negative in assays for primary 5' ends. Nevertheless, there seem to be a multitude of mRNA 5' ends and also 3' termini that are generated by processing events. Almost nothing is known about the enzymes and specificities involved in the generation of these termini, which most likely include endo- as well as exonucleolytic activities. Much more is known about processing of structural RNA molecules, especially of tRNAs, where endonucleolytic cuts are involved in the generation of mature 5' and 3' ends (see below).

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B. Intron Splicing in Mitochondrial Transcripts A number of introns have been identified to interrupt various mitoehondrial genes. In Arabidopsis thaliana, for example, the had1, 2, 4, 5, 7, rpl2, rps3, ccb452, and cox2 genes contain one to four introns each (1). The mitoehondrial introns in higher plants are almost exclusively group II introns, classified by their typical secondary structure (78). Only one group I intron has been identified as a phylogenetically recent invader (79). In other groups of land plants such as ferns and mosses several group II introns have been found as homologs to the seed plant introns, but the absence of further complete genome sequenees preclude any statement with respect to the presence of other introns, for example, of group I (80). In the liverwort Marchantia polymorpha, however, the complete sequence analysis of the mitochondrial genome identified several group II as well as seven group I introns, suggesting that this type of intron could well be present in other lineages of land plants (81). Splicing of group II introns includes two transesterification reactions and results in the formation of a lariat structure of the excised intron. Besides the usual cis arrangements with all exons and introns being part of a single gene or transcript, respectively, several trans-splicing introns have been described in plant mitochondria. These are exclusively found in the had1, had2, and nad5 genes and are usually interrupted in the highly variable domain IV of the intron (82-84). The mature mRNA must be formed by cis- and trans-splicing events from several independent RNA precursor molecules, which are encoded in completely different genomic regions within the chondriome. Interestingly the number of the trans-splicing introns found in nadl genes varies in different higher plant species, Oenothera containing two trans introns and wheat and Petunia containing three (85-87). Investigations of the origin of the mitochondrial trans introns in land plants revealed that they are derived from "normal" cis arrangements, which can be still found in different lower land plant lineages (80). Trans-splieing introns had initially been identified in chloroplasts of algae and plants (88-90). In Chlamydomonas reinhardtii plastids one trans intron in the psaA gene is even eomposed of three different RNA moieties with the third RNA molecule, an internal intron fragment (tscA), being encoded by an independent locus without any neighboring exon sequences (91). An analogous arrangement has also been suggested for the third intron of the had5 gene in Oenothera, where one part of this intervening sequenee has apparently been translocated to a different independent genomic location (92). Variation has also been observed in the number of cis introns present in the same gene in different plant species. The cox2 gene, for example, can contain either no intron (Oenothera) or one (maize) or two intervening sequences (carrot) (93-95).

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For plant mitochondria no in vitro splicing system is available that could help to identify the proteins involved in splicing of this group of introns. In Chlamydomonas reinhardtii a number of nuclear mutants have been described that are impaired in chloroplast group II intron splicing, and nuclear mutants affecting group II intron excision have also been identified for maize chloroplasts (96-99). Perhaps the complete sequence of the nuclear genome ofArabidopsis thaIiana will reveal some candidate genes for potential splicing factors.

C. Messenger RNA Processingand Stability As mentioned in earlier sections, the steady-state levels of plant mitochondrial RNAs are most likely determined by a combination of transcriptional rates and the stability of the transcripts. The latter is influenced by posttranscriptional processes, whose modes and processes have been extensively investigated in bacteria and in the cytoplasm of eukaryotes (100, 101 ). Numerous studies have investigated mRNA maturation and RNA stability in organelles of eukaryotic cells, for example, in mitochondria of animals and yeast and in chloroplasts of higher plants as well as in Chlamydomonas reinhardtii (102, 103). A common feature of these processes is the involvement of RNA secondary structure elements, which function as processing signals and stability elements. In bacteria and chloroplasts in higher plants and in Chlamydomonas reinhardtii, RNA stem loop structures, encoded by inverted repeats, are present in the 3' untranslated regions of many genes. In bacteria and similarly also in chloroplasts of higher plants a protein complex binds to such secondary structures. This complex contains several proteins, including endo- and exonucleases, which in a combined action are involved in maturation as well as in the final degradation of mRNAs. Differing compositions of these complexes most likely determine the resulting half-life of an mRNA. Interestingly, some of the constituents of these complexes are homologous proteins in bacteria and chloroplasts, for example, the bacterial PNPase and the PNPase-like 100-kDa protein of spinach. This similarity highlights the eubacteria-like origin of the framework controlling mRNA stability in this organelle (104-108). While in chloroplasts, the function of the stem loop structures seems to be restricted to mRNA processing and stability, in prokaryotes such inverted repeats might have dual functions in transcription termination as well as in RNA stability. Similar inverted repeats have also been identified in the 3' nontranslated regions of several plant mitochondrial genes, and the localization of 3' mRNA termini immediately downstream of these structures raised the possibility that they function analogously to the structures in bacteria involved in transcription termination and/or mediation of RNA stability (109). The presence or absence of such stem loop structures in otherwise almost identical cob transcripts correlates with differing steady-state abundances in certain rice and wheat lines, suggesting a function of these stem loops in transcript stabilization (110, 111 ). The 3' regions

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of the Ogura-type cytoplasmic male sterility-related orf138 mRNAs are likewise involved in determining their stabilities in three different Brassica hybrids (112) (details are presented in Section V). To study the function of the double stein loops in plant mitochondria, two different in vitro systems were used to differentiate between the potential transcription termination or mRNA stability functions. Investigations of the pea atp9 inverted repeat in a homologous in vitro transcription system indicated that transcription proceeds through this structure, so that a function as transcription terminator is highly unlikely (36). Analogous results were obtained with respective structures located downstream of the Oenothera atpl gene and the rice cob-1 gene, which substantiates that such stem loop structures do not function as plant mitochondrial transcription terminators at least in vitro (j. Kuhn and S. Binder, unpublished results). The functional analysis of the pea double stern loop in an in vitro processing system, however, demonstrated its involvement as a processing signal and stability element. RNA precursor molecules extending beyond the double stem loop structure are correctly processed with 3' ends that coincide with those mapped in vivo. In addition, an increased half-life of an RNA with stem loop compared to transcripts without such a structure confirmed the stabilizing effect of such stem loops. Thus, similar to the analogous structures in chloroplasts, plant mitochondrial inverted repeats seem to act as mRNA processing signals and stability elements rather than transcription terminators. In contrast to chloroplasts, where several components of the processing complex have been identified and characterized, virtually nothing is known about the constituents of the RNA processing machinery in plant mitochondria. The recent identification of an RNA helicase SUV3 in Arabidopsis thaliana is a first step toward the identification and characterization of these proteins (113). The SUV3 protein in yeast mitochondria is part ofa degradosome-like complex that includes also an exoribonuclease and may control mRNA stability in these organelles. However, the function of this RNA helicase in plant mitochondria remains to be determined since RNA processing in mitochondria of plants and yeast seems to be different (114-116). Beside these stabilizing processes there are also degradation-promoting mechanisms operative in plant mitochondria. Recently the degradation of CMSrelated atpl-orf522 transcripts in sunflower was shown to be correlated with preferential polyadenylation followed by degradation by a ribonuclease activity which preferentially degrades polyadenylated RNAs (117; further details see below). Shortly later polyadenylation of cox2 transcripts was reported in maize mitochondria, where nonencoded adenosines were found to be added at multiple sites throughout the transcript, with two major sites coinciding with mapped 3' ends. As in the case of the atpl-orf522 transcripts, polyadenylation seems to destabilize the maize cox2 transcripts in vitro, although the observed effect

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is very weak. Poly(A) tails were found at transcripts showing differing RNA editing stages, suggesting polyadenylation and RNA editing to be independent processes (118). Although polyadenylation seems to accelerate degradation of RNA in plant mitoehondria, its effect is quite weak in comparison to its significant destabilizing effect in bacteria and chloroplasts (119-121). In pea mitochondria, poly(A) tails were detected at the 3' ends of atp9 transcripts. They are attached at sites located within the second stem or just downstream coinciding with previously mapped 3' ends. Thus the stabilizing effect of the double stem loops and the degradation-promoting poly(A) tail counteract in a single mRNA. In vitro processing assays with transcripts containing a poly(A) tail downstream of the double stem loop revealed an accelerated processing of the RNAs compared to molecules without such a poly(A) extension but no significant enhancement of total degradation of the RNA. Thus the stabilizing effect of the stem loop seems to be stronger. The enhanced removal of the poly(A) sequences downstream of the stem loop may, however, help to remove the stem loop structure step by step in several consecutive rounds of polyadenylation and poly(A) removal. This model is supported by the distribution of the poly(A) attachment sites throughout the stem loop (122) (Fig. 3). The approaches to identify poly(A) sequences at plant mitochondrial transcripts described above all engage an RT-PCR analysis using an oligo(dT)adapter primer. These analyses thus have a bias toward the identification of noneneoded adenosines. To circumvent this we have recently used an alternative approach in which an anchor primer was directly ligated to the RNA followed by RT-PCR analysis from a primer complementary to the anchor. This approach confirmed the presence of nonencoded nucleotides at the 3' ends of pea atp9 transcripts, but surprisingly identified a large number of eytidines besides the adenosines in the only maximally three-nucleotide-long extensions. The composition of these short tracts including a perfect 5'-CCA-3' triplet indicates that they may be generated by the tRNA terminal nueleotidyl transferase (122). This observation allows several speculations about the generation of these noneneoded extensions at plant mitoehondrial transcript termini. First, there may be two different activities present, one that adds longer tails preferentially composed of adenosines and a second that adds short tails preferentially composed of adenosines and eytidines, possibly by the tRNA enzyme mentioned above. While the longer poly(A) tails clearly seem to support degradation, the effect of the short extensions is unclear. It may also be possible that the short extensions are remnants of larger extensions that have not been completely removed. In extrapolation it is possible that longer tails composed of other nueleotides (C, U, G) exist which were not detected by the approach with the oligo(dT)-adapter primer and remain difficult to detect since they might be

A

B

GGT ACG G T C G GAG~c GG A~T

g

Transcription Continues Through Inverted Repeats

A--T

G--C A~T A~T

GTA C

~

Acc:e~lo,~Pmmin(s)

A~T G~C AC'i-i-rCG]TCrGGAG G 5' (N) GGACGAGGC--G C--G C--G C--G C--G A--T G A CA

T ACT'Ir'rC G ~ 5' (HI G G A C G ~

Exorlbonuclease

A--T G A CA

D

C

GTA C

GGTA CG G T C G AG~ G

r

Processed 3' E nd

~G

~CAC 5' (N) G G A C G ~

A--T Gc AA

A--T G AA C

F

E GGTA CG G T C G

GGTA CG U C T G

A-Ai

~,

5' (N) G G A C G ~ A--T GC AA

of Polyadenylatlon and Progresslve Degradation

~

: A-G ~ AAAAAAAAAAA

5'

A--T G A CA

FIG. 3. Processing and degradation at the 3' termini of pea atp9 transcripts. Detailed in vitro studies of pea atp9 mRNA processing suggest the following model. (A) Transcription runs through the double inverted repeat and terminates at an as-yet-unknown termination structure. (B) Several proteins with potential stabilizing function bind to the double-stern-loop structure. These could also include the mitochondrial RNA helicase (SUV3), which has been identified as a component of a degradosome-like complex in yeast. Sequences downstream of the stem loop are removed most likely by an exoribonucleolytic activity. (C) Processed 3' ends surrounding the 3' end of the second stem are formed. (D) Nonencoded nucleotides, predominantly adenosines, are posttranscriptionally added to the 3' end by a poly(A) polymerase-like activity, rendering the mRNA more attractive for exonucleolytic digestion. (E) Again, downstream moieties are removed by an exoribonuclease proceeding several nucleotides into the stem loop structure. (F) This RNA is now again polyadenylated followed again by exoribonucleolytic digestion. Most likely several cycles of polyadenylation and digestion finally result in the removal of the stabilizing stem loop structure and the final total degradation of the atp9 mRNAs.

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comparatively rare and can be removed very rapidly similar to the poly(A). If these tails are generated by a single enzyme activity, its identity will indeed be very interesting.

D. RNA Editing by Base Modification Among the RNA processing and modification features so far described, RNA editing is certainly one of the most unusual. Initially identified in mitochondria oftrypanosomes, where nonencoded uridines are added or encoded uridines are deleted from an RNA posttranscriptionally (123), various types of RNA editing have been described in many different organisms. In general the term RNA editing describes processes that alter the information content of RNA so that the sequence of the edited RNA differs from the sequence encoded in the DNA. RNA editing is mechanistically classified into two major groups: (1) processes that insert or excise nucleotides from an RNA precursor and (2) reactions where a nucleotide identity is modified within the RNA molecule, for example, by deamination. RNA editing in plant mitochondria, discovered in 1989, is a typical "modificational" editing, which converts cytidines into uridines or in rare cases uridines to cytidines. Several studies indicate that the sugar-phosphate backbone of the RNA remains intact during this conversion, which seems to be accomplished by deamination or transamination reactions (123-125). Recently the screening of the transcripts of all known genes and of the well-conserved open reading frames encoded in the mitochondrial genome of Arabidopsis thaliana revealed a total number of 456 editing sites. These are exclusively C-to-U conversions, of which 441 are observed within coding regions. The process affects individual coding regions with frequencies varying from 0% to almost 19% of the eodons. Probably incidentally as a side effect of the codon distribution RNA editing increases the hydrophobicity of the proteins encoded (126). Further details of RNA editing in plant mitochondria have recently been reviewed and discussed extensively (124, 125).

IV. Processingof tRNAs and rRNAs A. A Variegated tRNA Population in Mitochondria of Higher Plants Translation in higher plant mitochondria follows the standard genetic code and thus requires a minimum of 23 different tRNAs considering the extended wobble and the separate initiator and elongator tRNA Met. Most of the tRNAs are encoded in the mitochondrial genome, but some are encoded by nuclear genes a n d have to be translocated into the organelle (127). In Arabidopsis thaliana

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the 22 tRNA genes in the mitoehondrial genome are not sufficient to decode the entire set of codons, and tRNAs for six amino acids have to be imported from the cytoplasm (1). In the mitochondrial DNA of sugar beet 25 tRNA genes are encoded and tRNAs for five amino acids have to be delivered from the cytosol (2). The diversity within the plant mitochondrial tRNA population is further increased by the different origins of the organelle-encoded tRNA genes, which are either of native (or genuine) mitochondrial origin derived from the original endosymbiont, or of chloroplast origin as parts of promiscuous chloroplast DNA sequences integrated into the plant mitochondrial DNA. A comprehensive study of the total plant mitochondrial tRNA population in potato revealed 31 different tRNAs, which are sufficient to decode all sense codons. Twenty of these, 15 native and 5 chloroplast-like, are encoded in the mitochondrion and 11, including 1 tRNAAla, 2 tRNAArg, 1 tRNA I/e, 5 tRNA Leu, and 2 tRNAThr, are nuclear-encoded (128). Although there are several nuclearencoded tRNAs that are imported in all plant species investigated, there are striking differences among the imported tRNAs of individual plant species. The set of imported tRNAs varies even within the angiosperm lineage, where, for example, the usually mitochondrially encoded tRNA ser (GCU) and tRNA ser (UGA) are imported into sunflower mitochondria (129). Interestingly, mitochondria of the liverwort Marchantia polymorpha contain different species of tRNAvaI from both nuclearly (AAC) and mitochondrially (UAC) encoded genes, although the latter would be sufficient to decode all four valine codons (GUN) (130). Little is known about the mechanisms of the tRNA import into plant mitochondria. There are no implications for sequence or secondary structure motifs that could specify or discriminate the set of nuclear-encoded mitochondrial tRNAs from the cytoplasmic tRNAs. This and the variable pattern of tRNA species imported in different plants suggest that each imported tRNA is recognized by a very specific factor. Candidate proteins for such specific mitochondrial carriers are the aminoacyl tRNA synthetases, and indeed an involvement ofalanyl tRNA synthetase has been indirectly substantiated in transgenic plants expressing a mutated tRNAAla. The Uz0-to-C70 mutation in this tRNA not only blocks aminoacylation, but also prevents the import of this tRNA into mitochondria (131,132). The exact role of these proteins in the import process is unclear. The dependence of the tRNA import on the presence of tRNA synthetases and on an intact protein import machinery has been shown in yeast, where two cytosolic lysine isoacceptor tRNAs are targeted to mitochondria (133, 134). In mitochondria of Trypanosoma brucei, however, where all mitochondrial tRNAs have to be imported from the cytoplasm, distinct pathways and mechanisms are suggested for the import of tRNAs and proteins. In this organism efficient tRNA import is observed in the absence of a membrane potential, which is an absolute prerequisite for protein translocation over the mitochondrial membrane. In addition, the lack of competition by protein precursors

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in the tRNA import suggests different receptor proteins on the mitochondrial surface (135). Thus, differing tRNA import mechanisms seem to exist in the different organisms investigated, which leaves it open whether an additional or an alternative pathway is used for tRNA import in plants. From the viewpoint of the endosymbiont theory, tRNA import is a newly developed mechanism, since neither of the potential participants of the symbiosis originally had a machinery to import or export ribonucleic acids. Thus, adaptation from a preexisting protein translocation system, as is suggested for at least some components of the protein import machinery in the chloroplast (136), seems possible, but may be so advanced that similarities to such proteins in existing relatives are no longer noticeable. A de novo development or an extensive modification with a retained flexibility might also be responsible for the observed differences between tRNA import machineries in different plant species.

B. Maturation of Mitochondrially Encoded tRNAs In general tRNAs have to undergo a series of processing steps until the mature molecule can fulfill its function in translation. The initial step in this maturation process in mitochondria of plants is usually the excision of the pre-mature tRNA from larger precursor molecules that were generated by transcription of the tRNA gene from upstream promoters (46-48) (Fig. 4). Competence for such a resection from precursor molecules has been confirmed by in vitro processing systems developed for wheat, Oenothera, potato, and pea (137-140). These in vitro analyses showed that the mature 5' ends of both native and chloroplastlike tRNAs are generated by exact endonucleolytic cleavages 5' to the discriminator nucleotides by an RNase P-like enzyme activity. As in bacteria, plant mitochondrial 5' processing seems to depend on both protein and RNA moieties, indicating that 5' processing in plant mitochondria is performed by a ribonucleoprotein. A similar enzyme structure has also been found in yeast, where the RNA subunit is encoded in the mitochondrion and the protein moiety is nuclear encoded (138, 141). Interestingly, mitochondrially encoded RNase P RNAs have also been found in Reclinomonas americana and in the green alga Nephroselmis olivacea, indicating that the homologous RNA in higher plants might also be encoded in the organelle (8, 142). Unfortunately, similarities between RNase P RNAs of different species are very low or simply nonexistent, effectively preventing identification of homologous RNAs by physical hybridization or computer analysis. The mature 3' end of plant mitochondrial tRNAs is formed by at least two enzyme activities. Respective in vitro systems showed that an endonuclease generates an intermediate 3' end which is identical with the 3' end of the tRNA gene. The 31 end is then further matured by the addition of a CCA triplet, which is not

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encoded in plant mitoehondrial tRNA genes. The respective tRNA nueleotidyl enzyme activity has been detected in mitochondrial protein lysates (137). The endonucleolytic 3' processing enzyme activity (called RNase Z) of plant mitochondria can be separated from RNase P activities, indicating that different enzymes are responsible for the two endonucleolytic reactions (143). In some instances RNase Z action requires a 5' processed precursor RNA, indicating and defining a clear order in which the 5' cut precedes 3' processing. The 3' maturation in plant mitochondria differs significantly from that in bacteria, where the CCA is encoded by the tRNA genes and the 3' end is generated by a series of endo- and exonucleolytic processing events (144). In the nuclear/cytosolic system in eukaryotes, however, endonucleolytic cleavage is, as in plant mitochondria, involved in tRNA 3' maturation. Despite the overt similarity, studies of both nuclear and mitochondrial activities revealed significant differences in the two cellular compartments, suggesting that these two enzymes are encoded by distinct genes (145). Especially in view of the modified endosymbiont theories, the nature of the mitochondrial 3' endonuclease will be very interesting. Besides the processing of the extremities of the tRNA, maturation also includes base modifications and in some instances RNA editing. Typical cytidine-to-uridine conversion has been found in tRNA Phe and tRNAcys from Oenothera and potato and in tRNA His from larch (146-148). In the acceptor stem of tRNA Phe editing corrects a C4-A69 mismatch to a U4-A69 Watson and Crick base pair. Three editing events likewise rescue correct base pairing in paired regions of tRNA His in larch. Conversely, the editing event detected in the anticodon stem of tRNA cys generates a noncanonical U2s-U42 pair, which is already present in the homologous tRNA from Marchantia, where no editing is found. A C2s is also present in prespermatophyta (Cycas) and primitive angiosperms (M agnoliidae), suggesting that this editing event is present in several lineages of land plants (149). Edited nucleotides are already seen in 5' and 3' extended precursor molecules, indicating that this processing precedes the cleavage reactions at the tRNA extremities (146). Indeed in vitro processing studies with edited and nonedited tRNA Phe and tRNA Hi~ precursor molecules showed that editing is a prerequisite for efficient 5' and 3' processing (Fig. 4). Thus the editing events can directly influence the amount of mature tRNA Phe or tRNA H~s " available in the total tRNA population (140, 148, 150). Interestingly, not all mismatches observed in acceptor stems of mitochondrial tRNAs are corrected by RNA editing, indicating that other parameters might be important to determine an editing event (151). A complex function can be assumed for the tRNA cy~ editing event. Here 5' and 3' processing, CCA addition, and aminoacylation are independent of the editing status of the tRNA molecule.

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MICHAELA HOFFMANN ET AL.

L

I

DNA Transcription i

Precursor RNA

i

CA

II

C RNA Editing

D

~[!Qsome" 3'

C ~'~ ~ 5' P rocessing RNase P

UA

C RNase Z

3' Processing

,O UA

C

II

D tRNA NucleoUdyltransferase

CCA Addition I I U A

C IL.O

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Two-dimensional analysis of the mature tRNA revealed a pseudo uridine at position 28, which has to be formed by a two-step reaction of deamination and isomerization and whose function is unclear (149). In summary, the generation of functional tRNAs is the best-characterized RNA maturation process in plant mitochondria. In contrast, almost nothing is known about the maturation processes of rRNAs. 18S and 5S rRNAs are generally part of an at least dicistronic precursor molecule whose transcription is initiated upstream of the mature 5' end of the 18S rRNA (29, 43, 152). Upstreamlocated promoters have also been detected for the 26S rRNA, although the detection of a 5' terminus accessible to in vitro capping of this RNA suggests that transcription is also initiated directly at the mature 5' end in potato (45). Nevertheless the documentation of 5' and 3' extended precursor molecules requires posttranscriptional processes to generate the mature extremities of these rRNAs. Since no in vitro processing systems are available for rRNA processing, one can only speculate about the processes and mechanisms involved in rRNA maturation. In wheat, for example, the 5' end of the 18S rRNA is generated by 3' processing of the tRNA fMet,whose 3' end lies only one nucleotide upstream of the 5' end of the rRNA. In addition a t-element (tRNA-like structure) that is located downstream of the 5S rRNA is efficiently processed by wheat mitochondrial protein lysates in vitro and may thus be involved in the maturation of this rRNA molecule in vivo (153). Although 5'-to-3' exonucleolytic digestion cannot be excluded, it is more likely that 5' ends are formed by an endonucleolytic cleavage. Generation of the 3' ends, on the other hand, could just as well involve both or either exo- and endonucleolytic activities.

V. Posttranscriptional RNA Processing Involved in Cytoplasmic Male Sterility Cytoplasmic male sterility (CMS) is a maternally inherited trait which interferes with the production of mature pollen. CMS has been observed in many different plant species and is usually associated with species-specific chimeric open reading frames in the mitochondrial DNA. The gene products most likely lead to mitochondrial dysfunction resulting in severe defects in male generative organs and thus preventing pollen development. CMS can be suppressed by

FIG. 4. Maturation oftBNA phe in plant mitochondria. After transcription of the tRNA precursor, which is initiated at an upstream promoter, RNA editing rescues a U - A canonical base pair from a mismatched C - A pair. This is a prerequisite for the 5 ~ and 3 r endonueleolytic cleavage reactions which generate the mature 5 ~ end and a pre-mature 3' end, and that is further modified by the addition of the 5'-CCA-3 t triplet.

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nuclear restorer genes via various mechanisms. These CM S-restorer systems can be used to produce hybrid seed and are thus of considerable commercial interest (154). This section will focus on selected examples where restoration of C MS is known to be mediated by altered transcript levels of the CMS-related ORFs. One of the best-characterized systems is that of Texas or T-cytoplasmic male sterility in maize. It has been attributed to expression of the T-urfl3 mosaic reading frame encoding a 13-kDa hydrophobic protein, which assembles to a tetrameric pore in the inner mitochondrial membrane (155). In mitochondria of the T-cytoplasm seven transcripts of T-urf13 and a cotranscribed orf221 are detected. Among these are 2.0-, 1.85-, 1.5-, and 1.0-kb-long mRNAs, which are, with the exception of the cappable 1.85-kb transcript, probably all generated by processing from a 3.8-kb RNA. Several restorer genes (Rf) independently introduce a new processing event, which reduces the amount of most of these transcripts. In the presence of the restorer genes Rfl, Rf8, and Rf* the T-urf13 transcript pattern is significantly altered with the 5' ends of novel transcripts, which are most likely generated from the 2.0-, 1.85-, 1.8-, and 1.0-kb long RNAs, located within the T-urf13 reading frame (156). Sequence conservation of these Rf-related 5' ends indicates that the three different restorer genes could encode or influence similar proteins responsible for this same specific alteration of the mRNAs. As a consequence of these altered transcript patterns, accumulation of the T-urf13 protein is reduced in the restored lines (155). In Brassica napus analogous alterations of the transcript patterns of two CMS-related genes designated orf224 and orf222 responsible for the so-called pol and nap CMS forms are observed upon restoration with respective restorer genes. In mitochondria of the pol cytoplasm transcripts of 2.2, 1.9, and 1.1 kb are generated from the atp6-orf224 region with the larger two RNAs spanning both genes. In plants restored with the pol-specific restorer gene Rfp two additional transcripts of 1.4 and 1.3 kb representing only the atp6 reading frame are observed and the 5' ends of these RNAs are located within the orf224 reading frame probably interfering with the generation of the respective protein from this ORF (157). Similarly an additional, smaller transcript is detected upon restoration of the nap-type CMS. The presence of the additional transcript perfectly cosegregates with the respective Rf genes, substantiating that the Rf gene product is somehow responsible for the generation of these altered transcripts. Interestingly, Rfp affects transcripts of two other mitochondrial regions encoding the had4 and a pseudogene of the cytochrome biogenesis gene ccbl, which are not correlated with CMS. In vitro 5' capping experiments further suggested the additional transcripts detected in restored pol plants to be generated by processing from the 2.2- and 1.9-kb transcripts rather than by de novo transcription initiation. It is unclear, however, whether an endo- or an exonucleolyticdigest gives rise to the Rf-specific transcripts (158). An endonucleolytic cleavage within transcripts of the CMS-associated orfl07 in Sorghum may

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analogously be responsible for restoration of the CMS phenotype in this plant species (159). The stability of RNA is also involved in the cytoplasmic male sterility of some Brassica cybrids. Here the CMS-related orf138 RNA is present in different configurations in three different cybrids. Transcripts of the BamHI/18S configuration (sterile) are 10-fold more abundant than those from the Nco2.5/13S (sterile) arrangement. This RNA abundance correlates with the presence of secondary structures in the 3' regions of these mRNAs and the translation of the orf138 protein responsible for CMS. In contrast no obvious secondary structure is present in the 3' region of the orf138 gene of the 13F cybrids (Nco2.7/13F) and accordingly no transcript is detected. As a result no orf138 gene product is generated correlating with fertility of this cybrid line. In vitro decay tests with synthetic transcripts representing the 3' region of the orf138 transcripts substantiated that the different RNA stabilities are responsible for the differing steady-state amounts of these CMS-related transcripts (112). CMS in sunflower is associated with the expression of a 15-kDa mitochondrial protein encoded by orf522. This open reading frame is cotranscribed with the upstream-located atpl gene on a 3.0-kb mRNA that is not detected in fertile plants. Detailed transcript analysis showed that atpl-orf522 transcripts are tissue- and cell-specifically reduced in the background of a restorer gene, which correlates with a reduced abundance of this protein in male florets (160, 161 ). Run-on experiments indicate that the lower steady-state level of this RNA is not due to a reduced transcriptional rate. A recent report shows that polyadenylation is involved in the tissue-specific reduction of the atpl-orf522 transcript. Male florets of fertility restored plants show an increase in the level of polyadenylatedatpl-orf522 transcripts correlated with tissue-specific reduction of this RNA in the steady-state. This is most likely mediated by enhanced degradation by an RNase activity that preferentially degrades polyadenylated transcripts (117), In summary, CM S and respective restorer gene systems show the importance of the stability of mitochondrial transcripts for the physiologic phenotype of the plant affected. These systems are thus potentially rewarding objects with which to study the mechanism of mRNA stability, processing, and degradation in plant mitochondria.

ACKNOWLEDGMENTS In this review we tried to give a coherent, up-to-date picture of plant mitochondrial gene expression. We apologize to all colleagues whose work could not adequately be referenced due to space limitations. Work in the authors' laboratory is supported by grants from the Deutsche Forschungsgemeinschft and the Fonds der Chemischen Industrie.

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