Despite the importance of pentatricopeptide repeat (PPR) proteins in organellar RNA metabolism and plant development, the functions of many PPR proteins remain unknown. Here, we determined the role ...of a mitochondrial PPR protein (At1g52620) comprising 19 PPR motifs, thus named PPR19, in Arabidopsis thaliana.
The ppr19 mutant displayed abnormal seed development, reduced seed yield, delayed seed germination, and retarded growth, indicating that PPR19 is indispensable for normal growth and development of Arabidopsis thaliana. Splicing pattern analysis of mitochondrial genes revealed that PPR19 specifically binds to the specific sequence in the 3′-terminus of the NADH dehydrogenase 1 (nad1) transcript and stabilizes transcripts containing the second and third exons of nad1. Loss of these transcripts in ppr19 leads to multiple secondary effects on accumulation and splicing of other nad1 transcripts, from which we can infer the order in which cis- and trans-spliced nad1 transcripts are normally processed.
Improper splicing of nad1 transcripts leads to the absence of mitochondrial complex I and alteration of the nuclear transcriptome, notably influencing the alternative splicing of a variety of nuclear genes.
Our results indicate that the mitochondrial PPR19 is an essential component in the splicing of nad1 transcripts, which is crucial for mitochondrial function and plant development.
Restorer-of-fertility (Rf) genes encode pentatricopeptide repeat (PPR) proteins that are targeted to mitochondria where they specifically bind to transcripts that induce cytoplasmic male sterility ...and repress their expression. In searching for a molecular signature unique to this class of proteins, we found that a majority of known Rf proteins have a distinct domain, which we called RfCTD (Restorer-of-fertility C-terminal domain), and its presence correlates with the ability to induce cleavage of the mitochondrial RNA target. A screen of 219 angiosperm genomes from 123 genera using a sequence profile that can quickly and accurately identify RfCTD sequences revealed considerable variation in RFL/RfCTD gene numbers across flowering plants. We observed that plant genera with bisexual flowers have significantly higher numbers of RFL genes compared to those with unisexual flowers, consistent with a role of these proteins in restoration of male fertility. We show that removing the RfCTD from the RFL protein RNA PROCESSING FACTOR 2-nad6 prevented cleavage of its RNA target, the nad6 transcript, in Arabidopsis thaliana mitochondria. We provide a simple way of identifying putative Rf candidates in genome sequences, new insights into the molecular mode of action of Rf proteins and the evolution of fertility restoration in flowering plants.
Since the endosymbiotic origin of chloroplasts from cyanobacteria 2 billion years ago, the evolution of plastids has been characterized by massive loss of genes. Most plants and algae depend on ...photosynthesis for energy and have retained ∼110 genes in their chloroplast genome that encode components of the gene expression machinery and subunits of the photosystems. However, nonphotosynthetic parasitic plants have retained a reduced plastid genome, showing that plastids have other essential functions besides photosynthesis. We sequenced the complete plastid genome of the underground orchid, Rhizanthella gardneri. This remarkable parasitic subterranean orchid possesses the smallest organelle genome yet described in land plants. With only 20 proteins, 4 rRNAs, and 9 tRNAs encoded in 59,190 bp, it is the least gene-rich plastid genome known to date apart from the fragmented plastid genome of some dinoflagellates. Despite numerous differences, striking similarities with plastid genomes from unrelated parasitic plants identify a minimal set of protein-encoding and tRNA genes required to reside in plant plastids. This prime example of convergent evolution implies shared selective constraints on gene loss or transfer.
The coding regions of many mitochondrial genes in plants are interrupted by intervening sequences that are classified as group II introns. Their splicing is essential for the expression of the genes ...they interrupt and hence for respiratory function, and is facilitated by various protein cofactors. Despite the importance of these cofactors, only a few of them have been characterized.
CRS1-YhbY domain (CRM) is a recently recognized RNA-binding domain that is present in several characterized splicing factors in plant chloroplasts. The Arabidopsis genome encodes 16 CRM proteins, but these are largely uncharacterized.
Here, we analyzed the intracellular location of one of these hypothetical proteins in Arabidopsis, mitochondrial CAF-like splicing factor 1 (mCSF1; At4 g31010), and analyzed the growth phenotypes and organellar activities associated with mcsf1 mutants in plants.
Our data indicated that mCSF1 resides within mitochondria and its functions are essential during embryogenesis. Mutant plants with reduced mCSF1 displayed inhibited germination and retarded growth phenotypes that were tightly associated with reduced complex I and IV activities. Analogously to the functions of plastid-localized CRM proteins, analysis of the RNA profiles in wildtype and mcsf1 plants showed that mCSF1 acts in the splicing of many of the group II intron RNAs in Arabidopsis mitochondria.
SUMMARY
The lycophyte Phylloglossum drummondii is the sole inhabitant of its genus in the Huperzioideae group and one of a small minority of plants which perform uridine to cytidine RNA editing. We ...assembled the P. drummondii chloroplast and mitochondrial genomes and used RNA sequence data to build a comprehensive profile of organellar RNA editing events. In addition to many C‐to‐U editing events in both organelles, we found just four U‐to‐C editing events in the mitochondrial transcripts cob, nad1, nad5 and rpl2. These events are conserved in related lycophytes in the genera Huperzia and Phlegmariurus. De novo transcriptomes for three of these lycophytes were assembled to search for putative U‐to‐C RNA editing enzymes. Four putative U‐to‐C editing factors could be matched to the four mitochondrial U‐to‐C editing sites. Due to the unusually few numbers of U‐to‐C RNA editing sites, P. drummondii and related lycophytes are useful models for studying this poorly understood mechanism.
Significance Statement
U‐to‐C RNA editing is only found in plants, and within plants, only in some hornworts, lycophytes and ferns. The unusual lycophyte Phylloglossum drummondii contains four U‐to‐C editing sites that can be matched with four putative U‐to‐C editing enzymes, making it a useful model for the study of this uncommon process.
Summary
RNA editing is an essential mechanism that modifies target cytidines to uridine in both mitochondrial and plastid mRNA. Target sites are recognized by pentatricopeptide repeat (PPR) proteins. ...Using bioinformatics predictions based on the code describing sequence recognition by PPR proteins, we have identified an Arabidopsis editing factor required for editing of atpF in plastids. A loss‐of‐function mutation in ATPF EDITING FACTOR 1 (AEF1, AT3G22150) results in severe variegation, presumably due to decreased plastid ATP synthase levels. Loss of editing at the atpF site is coupled with a large decrease in splicing of the atpF transcript, even though the editing site is within an exon and 53 nucleotides distant from the splice site. The rice orthologue of AEF1, MPR25, has been reported to be required for editing of a site in mitochondrial nad5 transcripts, and we confirm that editing of the same site is affected in the Arabidopsis aef1 mutant. We also show that splicing of chloroplast atpF transcripts is affected in the rice mpr25 mutant. AEF1 is thus highly unusual for an RNA editing specificity factor in that it has functions in both organelles.
Significance Statement
The RNA editing factor AEF1/MPR25 is a sequence‐specific RNA binding protein with an unusual multi‐functional role in editing and splicing of the chloroplast atpF transcript. It is unique (so far) amongst editing specificity factors in also acting in mitochondria, where it recognizes a similar sequence to induce editing of the nad5 transcript.
Mitochondrial genomes (mtDNAs) in angiosperms contain numerous group II‐type introns that reside mainly within protein‐coding genes that are required for organellar genome expression and respiration. ...While splicing of group II introns in non‐plant systems is facilitated by proteins encoded within the introns themselves (maturases), the mitochondrial introns in plants have diverged and have lost the vast majority of their intron‐encoded ORFs. Only a single maturase gene (matR) is retained in plant mtDNAs, but its role(s) in the splicing of mitochondrial introns is currently unknown. In addition to matR, plants also harbor four nuclear maturase genes (nMat 1 to 4) encoding mitochondrial proteins that are expected to act in the splicing of group II introns. Recently, we established the role of one of these proteins, nMAT2, in the splicing of several mitochondrial introns in Arabidopsis. Here, we show that nMAT1 is required for trans‐splicing of nad1 intron 1 and also functions in cis‐splicing of nad2 intron 1 and nad4 intron 2. Homozygous nMat1 plants show retarded growth and developmental phenotypes, modified respiration activities and altered stress responses that are tightly correlated with mitochondrial complex I defects.
Although chloroplast transformation is possible in some plant species, it is extremely difficult to create or select mutations in plant mitochondrial genomes, explaining why few genetic studies of ...mitochondrially encoded functions exist. In recent years it has become clear that many nuclear genes encode factors with key functions in organelle gene expression, and that often their action is restricted to a single organelle gene or transcript. Mutations in one of these nuclear genes thus leads to a specific primary defect in expression of a single organelle gene, and the nuclear mutation can be used as a surrogate for a phenotypically equivalent mutation in the organelle genome. These surrogate mutations often result in defective assembly of respiratory complexes, and lead to severe phenotypes including reduced growth and fertility, or even embryo-lethality. A wide collection of such mutants is now available, and this review summarises the progress in basic knowledge of mitochondrial biogenesis they have contributed to and points out areas where this resource has not been exploited yet.
Summary
The pentatricopeptide repeat proteins PPR4 and EMB2654 have been shown to be required for the trans‐splicing of plastid rps12 transcripts in Zea mays (maize) and Arabidopsis, respectively, ...but their roles in this process are not well understood. We investigated the functions of the Arabidopsis and Oryza sativa (rice) orthologs of PPR4, designated AtPPR4 (At5g04810) and OsPPR4 (Os4g58780). Arabidopsis atppr4 and rice osppr4 mutants are embryo‐lethal and seedling‐lethal 3 weeks after germination, respectively, showing that PPR4 is essential in the development of both dicot and monocot plants. Artificial microRNA‐mediated mutants of AtPPR4 displayed a specific defect in rps12 trans‐splicing, with pale‐green, yellowish or albino phenotypes, according to the degree of knock‐down of AtPPR4 expression. Comparison of RNA footprints in atppr4 and emb2654 mutants showed a similar concordant loss of extensive footprints at the 3′ end of intron 1a and at the 5′ end of intron 1b in both cases. EMB2654 is known to bind within the footprint region in intron 1a and we show that AtPPR4 binds to the footprint region in intron 1b, via its PPR motifs. Binding of both PPR4 and EMB2654 is essential to juxtapose the two intron halves and to maintain the RNAs in a splicing‐competent structure for the efficient trans‐splicing of rps12 intron 1, which is crucial for chloroplast biogenesis and plant development. The similarity of EMB2654 and PPR4 orthologs and their respective binding sites across land plant phylogeny indicates that their coordinate function in rps12 trans‐splicing has probably been conserved for 500 million years.
Significance Statement
Despite the crucial role of pentatricopeptide repeat (PPR) proteins in organellar RNA metabolism and plant development, the functions of many PPR proteins remain unknown. Here, we show that the PPR4 and EMB2654 bind to the 5′ end of rps12 intron 1b and the 3′ end of rps12 intron 1a to juxtapose the two intron halves for the efficient trans‐splicing of rps12 intron 1, which is essential for chloroplast biogenesis and plant development.
Summary
Mitochondrial biogenesis relies on nuclearly encoded factors, which regulate the expression of the organellar‐encoded genes. Pentatricopeptide repeat (PPR) proteins constitute a major gene ...family in angiosperms that are pivotal in many aspects of mitochondrial (mt)RNA metabolism (e.g. trimming, splicing, or stability). Here, we report the analysis of MITOCHONDRIA STABILITY/PROCESSING PPR FACTOR1 (MSP1, At4g20090), a canonical PPR protein that is necessary for mitochondrial functions and embryo development.
Loss‐of‐function allele of MSP1 leads to seed abortion. Here, we employed an embryo‐rescue method for the molecular characterization of msp1 mutants.
Our analyses reveal that msp1 embryogenesis fails to proceed beyond the heart/torpedo stage as a consequence of a nad1 pre‐RNA processing defect, resulting in the loss of respiratory complex I activity. Functional complementation confirmed that msp1 phenotypes result from a disruption of the MSP1 gene.
In Arabidopsis, the maturation of nad1 involves the processing of three RNA fragments, nad1.1, nad1.2, and nad1.3. Based on biochemical analyses and mtRNA profiles of wild‐type and msp1 plants, we concluded that MSP1 facilitates the generation of the 3′ terminus of nad1.1 transcript, a prerequisite for nad1 exons a–b splicing. Our data substantiate the importance of mtRNA metabolism for the biogenesis of the respiratory system during early plant life.
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