Gene Expression In Plant Mitochondria Research Articles

The P-type pentatricopeptide repeat protein DWEORG1 is a non-previously reported rPPR protein of Arabidopsis mitochondria

Gene expression in plant mitochondria is mainly regulated by nuclear-encoded proteins on a post-transcriptional level. Pentatricopeptide repeat (PPR) proteins play a major role by participating in mRNA stability, splicing, RNA editing, and translation initiation. PPR proteins were also shown to be part of the mitochondrial ribosome (rPPR proteins), which may act as regulators of gene expression in plants. In this study, we focus on a mitochondrial-located P-type PPR protein—DWEORG1—from Arabidopsis thaliana. Its abundance in mitochondria is high, and it has a similar expression pattern as rPPR proteins. Mutant dweorg1 plants exhibit a slow-growth phenotype. Using ribosome profiling, a decrease in translation efficiency for cox2, rps4, rpl5, and ccmFN2 was observed in dweorg1 mutants, correlating with a reduced accumulation of the Cox2 protein in these plants. In addition, the mitochondrial rRNA levels are significantly reduced in dweorg1 compared with the wild type. DWEORG1 co-migrates with the ribosomal proteins Rps4 and Rpl16 in sucrose gradients, suggesting an association of DWEORG1 with the mitoribosome. Collectively, this data suggests that DWEORG1 encodes a novel rPPR protein that is needed for the translation of cox2, rps4, rpl5, and ccmFN2 and provides a stabilizing function for mitochondrial ribosomes.

Pentatricopeptide repeat protein MITOCHONDRIAL STABILITY FACTOR 3 ensures mitochondrial RNA stability and embryogenesis.

Gene expression in plant mitochondria is predominantly governed at the post-transcriptional level and relies mostly on nuclear-encoded proteins. However, the protein factors involved and the underlying molecular mechanisms are still not well understood. Here, we report on the function of the MITOCHONDRIAL STABILITY FACTOR 3 (MTSF3) protein, previously named EMBRYO DEFECTIVE 2794 (EMB2794), and show that it is essential for accumulation of the mitochondrial NADH dehydrogenase subunit 2 (nad2) transcript in Arabidopsis (Arabidopsis thaliana) but not for splicing of nad2 intron 2 as previously proposed. The MTSF3 gene encodes a pentatricopeptide repeat protein that localizes in the mitochondrion. An MTSF3 null mutation induces embryonic lethality, but viable mtsf3 mutant plants can be generated through partial complementation with the developmentally regulated ABSCISIC ACID INSENSITIVE3 promoter. Genetic analyses revealed growth retardation in rescued mtsf3 plants owing to the specific destabilization of mature nad2 mRNA and a nad2 precursor transcript bearing exons 3 to 5. Biochemical data demonstrate that MTSF3 protein specifically binds to the 3' terminus of nad2. Destabilization of nad2 mRNA induces a substantial decrease in complex I assembly and activity and overexpression of the alternative respiratory pathway. Our results support a role for MTSF3 protein in protecting two nad2 transcripts from degradation by mitochondrial exoribonucleases by binding to their 3' extremities.

The translational landscape of Arabidopsis mitochondria.

Messenger RNA translation is a complex process that is still poorly understood in eukaryotic organelles like mitochondria. Growing evidence indicates though that mitochondrial translation differs from its bacterial counterpart in many key aspects. In this analysis, we have used ribosome profiling technology to generate a genome-wide snapshot view of mitochondrial translation in Arabidopsis. We show that, unlike in humans, most Arabidopsis mitochondrial ribosome footprints measure 27 and 28 bases. We also reveal that respiratory subunits encoding mRNAs show much higher ribosome association than other mitochondrial mRNAs, implying that they are translated at higher levels. Homogenous ribosome densities were generally detected within each respiratory complex except for complex V, where higher ribosome coverage corroborated with higher requirements for specific subunits. In complex I respiratory mutants, a reorganization of mitochondrial mRNAs ribosome association was detected involving increased ribosome densities for certain ribosomal protein encoding transcripts and a reduction in translation of a few complex V mRNAs. Taken together, our observations reveal that plant mitochondrial translation is a dynamic process and that translational control is important for gene expression in plant mitochondria. This study paves the way for future advances in the understanding translation in higher plant mitochondria.

Comparison of mitochondrial gene expression and polysome loading in different tobacco tissues

BackgroundTo investigate translational regulation of gene expression in plant mitochondria, a mitochondrial polysome isolation protocol was established for tobacco to investigate polysomal mRNA loading as a proxy for translational activity. Furthermore, we developed an oligonucleotide based microarray platform to determine the level of Nicotiana tabacum and Arabidopsis thaliana mitochondrial mRNA.ResultsMicroarray analysis of free and polysomal mRNAs was used to characterize differences in the levels of free transcripts and ribosome-bound mRNAs in various organs of tobacco plants. We have observed higher mitochondrial transcript levels in young leaves, flowers and floral buds as compared to fully expanded leaves and roots. A similar pattern of abundance was observed for ribosome-bound mitochondrial mRNAs in these tissues. However, the accumulation of the mitochondrial protein COX2 was found to be inversely related to that of its ribosome-bound mRNA.ConclusionsOur results indicate that the association of mitochondrial mRNAs to ribosomes is largely determined by the total transcript level of a gene. However, at least for Cox2, we demonstrated that the level of ribosome-bound mRNA is not reflected by the amount of COX2 protein.

In vitro RNA uptake studies in plant mitochondria.

During evolution, most of the ancestral genes from the endosymbiotic α-proteobacteria at the origin of mitochondria have been either lost or transferred to the nuclear genome. To allow the comeback of proteins and RNAs [in particular transfer RNA (tRNAs)] into the organelle, macromolecule import systems were universally established. While protein import processes have been studied into details, much less is known about tRNA mitochondrial import. In plants, part of the knowledge on the tRNA import process into mitochondria has been acquired thanks to in vitro import assays. Furthermore, the development of in vitro RNA import strategies allowed the study of plant mitochondrial gene expression. The purpose of this chapter is to provide detailed protocols to perform in vitro RNA uptake into potato (Solanum tuberosum) or Arabidopsis (Arabidopsis thaliana) mitochondria as well as approaches to analyze them.

Post-transcriptional Regulation in Mitochondria of Rice and Wheat at Low Temperatures

Land plants respond to cold through complicated physiological, morphological, and metabolic pro- cesses, including abnormalities and changes in organelles, while plant mitochondrial gene expres- sions are regulated through several unique post-transcriptional events, such as cis-/trans-intron splic- ing and RNA editing. The author tries to clarify the relationship between these post-transcriptional events at low temperatures. Some intron-containing transcripts before splicing increase in rice and wheat after several days under cold conditions, while certain RNA editing events in IBS (Intron Binding Sites: which is present in exons and make complementary base pairing with Exon Binding Sites located in introns) have tight associations with splicing, some of which are sensitive to cold in intron-containing transcripts. However no correlation is observed between post-transcriptional events and the organization of introns: the primary sequence of introns, splicing manner (cis or trans), intron length, and splice-site sequence. These findings suggest that nucleus-encoded factors regulate mitochondrial gene expressions, some of which are cold-sensitive. In wheat, a cold acclimatable plant, accumulation of some intron-spliced transcripts also increase at low temperatures, as well as intron-containing transcripts, which may be one of the phenomena related to cold acclimation. The study about plant mitochondrial gene expression at low temperatures has the potential to become an alternative system to see how mitochondrial genes are regulated, which, in turn, has the potential to enhance plant breeding for cold tolerance.

The pentatricopeptide repeat MTSF1 protein stabilizes the nad4 mRNA in Arabidopsis mitochondria

Gene expression in plant mitochondria involves a complex collaboration of transcription initiation and termination, as well as subsequent mRNA processing to produce mature mRNAs. In this study, we describe the function of the Arabidopsis mitochondrial stability factor 1 (MTSF1) gene and show that it encodes a pentatricopeptide repeat protein essential for the 3′-processing of mitochondrial nad4 mRNA and its stability. The nad4 mRNA is highly destabilized in Arabidopsis mtsf1 mutant plants, which consequently accumulates low amounts of a truncated form of respiratory complex I. Biochemical and genetic analyses demonstrated that MTSF1 binds with high affinity to the last 20 nucleotides of nad4 mRNA. Our data support a model for MTSF1 functioning in which its association with the last nucleotides of the nad4 3′ untranslated region stabilizes nad4 mRNA. Additionally, strict conservation of the MTSF1-binding sites strongly suggests that the protective function of MTSF1 on nad4 mRNA is conserved in dicots. These results demonstrate that the mRNA stabilization process initially identified in plastids, whereby proteins bound to RNA extremities constitute barriers to exoribonuclease progression occur in plant mitochondria to protect and concomitantly define the 3′ end of mature mitochondrial mRNAs. Our study also reveals that short RNA molecules corresponding to pentatricopeptide repeat-binding sites accumulate also in plant mitochondria.

Ribosomal Regulation of Mitochondrial Gene Expression

Several mitochondrial protein complexes contain subunits encoded in the nucleus as well as those encoded in mitochondria. Despite the essential nature of the few proteins encoded in mitochondria, surprisingly little is known about plant mitochondrial gene expression and its regulation ([Millar et al

Silencing of the Nuclear RPS10 Gene Encoding Mitochondrial Ribosomal Protein Alters Translation in Arabidopsis Mitochondria

Hardly anything is known about translational control of plant mitochondrial gene expression. Here, we provide evidence for differential translation of mitochondrial transcripts in Arabidopsis thaliana. We found that silencing of the nuclear RPS10 gene encoding mitochondrial ribosomal protein S10 disturbs the ratio between the small and large subunits of mitoribosomes, with an excess of the latter. Moreover, a portion of the small subunits are incomplete, lacking at least the S10 protein. rps10 cells also have an increased mitochondrial DNA copy number per cell, causing an upregulation of all mitochondrial transcripts. Mitochondrial translation is also altered so that it largely overrides the hyperaccumulation of transcripts, and as a consequence, only ribosomal proteins are oversynthesized, whereas oxidative phosphorylation subunits are downregulated. Expression of nuclear-encoded components of mitoribosomes and oxidative phosphorylation system (OXPHOS) complexes seems to be less affected. The ultimate coordination of expression of the nuclear and mitochondrial genomes occurs at the complex assembly level. These findings indicate that mitoribosomes can regulate gene expression by varying the efficiency of translation of mRNAs for OXPHOS and ribosomal proteins.

Two DEAD-Box Proteins May Be Part of RNA-Dependent High-Molecular-Mass Protein Complexes in Arabidopsis Mitochondria

Posttranscriptional processes are important for regulation of gene expression in plant mitochondria. DEAD-box proteins, which form a huge protein family with members from all kingdoms, are fundamental components in virtually all types of processes in RNA metabolism. Two members of this protein family, designated PMH1 and PMH2 (for PUTATIVE MITOCHONDRIAL RNA HELICASE), were analyzed and characterized in mitochondria of Arabidopsis (Arabidopsis thaliana). Green fluorescent protein tagging with N-terminal PMH1 and PMH2 sequences supports the mitochondrial localization of these proteins. Northern experiments, as well as histochemical beta-glucuronidase staining of transgenic plants carrying respective promoter:beta-glucuronidase fusion constructs, revealed differing transcription patterns for the two genes. In response to cold, however, transcript levels of both genes increased. Immunodetection analyses of mitochondrial protein complexes after two-dimensional blue native/urea SDS-PAGE and after fractionation on sucrose gradients strongly suggest that one or both proteins are part of RNA-dependent complexes. Cold treatment of cell cultures or solubilization of mitochondria in the presence of MgCl(2) favored the detection of high-molecular-mass complexes. This study paves the way for detailed analysis of high-molecular-mass complexes in mitochondria of higher plants.

The multifarious roles of PPR proteins in plant mitochondrial gene expression

With a few rare exceptions, genes encoding pentatricopeptide (PPR) proteins are present in all sequenced eukaryotic genomes but absent from prokaryotic and archaeal genomes. The family has greatly expanded in plants, to more than 400 genes in each species. So far, the evidence indicates that PPR proteins are generally involved in regulation of organelle genome expression, in other words they are eukaryotic proteins selected for the control of genomes of prokaryotic origin. PPR proteins are localised in both plastids and mitochondria, and appear to have similar roles in both cases. They have been implicated in almost all stages of gene expression, including messenger RNA (mRNA) transcription, splicing, processing, editing, translation and stability. The most probable hypothesis for explaining these diverse roles is that PPR proteins are sequence‐specific RNA‐binding adaptors capable of directing effector enzymes to defined sites on mRNAs. Much of the recent interest in the role of PPR proteins in mitochondria has been driven by the discovery that most cytoplasmic male sterility systems comprise fertility restorer genes that are members of this fascinating family.

Transcript levels in plant mitochondria show a tight homeostasis during day and night

In plants the physiological and biochemical demands on each cell vary greatly between day and night, mostly due to the differing output of photosynthesis. Chloroplasts, the organelles of photosynthesis, are biochemically closely linked to the other energy generating organelles, the mitochondria. We have now investigated whether gene expression in plant mitochondria is influenced by these daily physiological variations. Transcript synthesis in these organelles cycles in a diurnal rhythm, while steady state transcript levels do not vary between light and dark phases and are stable throughout the diurnal (as well as the circadian) time course. This finding suggests that available steady state transcript levels in plant mitochondria are sufficient to provide the required biochemical capacities also at times of peak respiratory and physiological demands.

Editing of the Mitochondrial cox3 Transcript May Yield a New Site of Protein–Protein Interactions in Wild Cereal Elymus sibiricus L.

With PCR, RT-PCR, and direct sequencing, complete nucleotide sequences were established for the Elymus sibiricus mitochondrial cytochrome c oxidase subunit 3 gene (cox3) and its cDNA. The cox3 transcript was shown to have 12 editing sites with changes affecting the amino acid sequence of the protein product. The editing of the primary cox3 transcript was found to change the position of a site of protein-protein interactions. The results demonstrate again the important role of mRNA editing in posttranscriptional regulation of the expression of plant mitochondrial genes.

Gene expression in plant mitochondria: transcriptional and post-transcriptional control.

The informational content of the mitochondrial genome in plants is, although small, essential for each cell. Gene expression in these organelles involves a number of distinct transcriptional and post-transcriptional steps. The complex post-transcriptional processes of plant mitochondria such as 5' and 3' RNA processing, intron splicing, RNA editing and controlled RNA stability extensively modify individual steady-state RNA levels and influence the mRNA quantities available for translation. In this overview of the processes in mitochondrial gene expression, we focus on confirmed and potential sites of regulatory interference and discuss the evolutionary origins of the transcriptional and post-transcriptional processes.

RT-PCR analysis of 5' to 3'-end-ligated mRNAs identifies the extremities of cox2 transcripts in pea mitochondria.

Gene expression in plant mitochondria is still inadequately analyzed. To learn more about transcription and RNA processing in plant mitochondria, the 5'- and 3'-RNA extremities and the promoters of the cytochrome oxidase gene (cox2) were analyzed in pea. Both 5' and 3' ends of cox2 transcripts were examined by RT-PCR across the ligation site of circularized mitochondrial RNA as template. This approach identified 5' ends a few nucleotides shorter than three major 5' ends mapped by primer extension analysis. Presumably, only monophosphate 5' ends derived from processing can be ligated. In vitro transcription assays using a homologous mitochondrial protein extract from pea strongly suggest the major 5' ends to derive from transcription initiation. The cDNA analysis of the head-to-tail ligated cox2 mRNA identified 3' ends within a thymidine stretch approximately 300 nt downstream of the reading frame in a sequence segment that was not present in the previous investigation of this gene. Nuclease S1 protection experiments confirmed this newly identified 3' terminus and corroborated the validity of this technique in mRNA end analysis. The general use of the circularized RNA (CR)-RT-PCR approach for the simultaneous analysis of the 5' and 3' extremities of mRNA molecules is discussed.

Gene expression in isolated plant mitochondria: high fidelity of transcription, splicing and editing of a transgene product in electroporated organelles

Mitochondrial gene expression was studied using an electrotransformation protocol to introduce foreign DNA into purified wheat mitochondria. Optimal conditions for DNA uptake and transient gene expression were determined. We show here that a DNA plasmid containing either a cognate or a non-cognate gene under the control of a plant mitochondrial promoter is incorporated into the organelle and faithfully recognized by the transcription machinery. Transcripts generated by a plasmid bearing the intron-containing cox II gene were correctly spliced. Moreover, the transcripts were edited at the expected target C residues. The expression and maturation process of the transgene is dependent on the integrity of functional elements such as the promotor or the presence of structural domains necessary for splicing. The mitochondrial transformation described in this report is an important tool to study the multiple steps involved in plant mitochondrial gene expression at conditions closer to those found in vivo.

The RNA world of plant mitochondria

Mitochondria are well known as the cellular power factory. Much less is known about these organelles as a genetic system. This is particularly true for mitochondria 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 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 mitochondria 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 3' 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.

RNA degradation buffers asymmetries of transcription in Arabidopsis mitochondria.

To understand better the relative contributions of transcriptional and post-transcriptional processes towards the regulation of gene expression in plant mitochondria, we compared the steady state levels of RNAs with the respective transcriptional activities. All of the protein and rRNA coding genes of the Arabidopsis mitochondrial genome and several orfs were analyzed by run-on and northern experiments. rRNAs constitute the bulk of the steady state RNA in Arabidopsis mitochondria, but are (different from maize mitochondria) not equally prominent among the run-on transcripts. Their relatively low rate of active transcription is apparently compensated by their high stability. Run-on transcription values differ significantly between genes coding for different subunits of the same protein complex. The steady state RNA levels are considerably more homogeneous, indicating that high variations of transcription rates are counterbalanced by post-transcriptional processes. The relative amounts of the steady state transcripts for the different subunits in a given protein complex reflect the relative stoichiometries of the protein subunits much more closely than the respective transcriptional activities. Post-transcriptional RNA processing and stability thus contribute significantly to the regulation of gene expression in Arabidopsis mitochondria.

Genomic context influences the activity of maize mitochondrial cox2 promoters.

Plant mitochondrial genomes are highly recombinogenic, with a variety of species-specific direct and inverted repeats leading to in vivo accumulation of multiple DNA forms. In maize, the cox2 gene, which encodes subunit II of cytochrome c oxidase, lies immediately downstream of a 0.7-kilobase direct repeat, which is present in two copies in the 570-kilobase master chromosome. Promoters for cox2 exist upstream of both of these copies, in regions we have termed A and B. Three region B promoters are active for cox2 transcription in the master chromosome, whereas two region A promoters are active for cox2 transcription after recombination across the direct repeats. We have measured the proportion of genomes carrying region A or B upstream of cox2 in maize seedlings and found a ratio of approximately 1:6. Promoter strength, based on run-on transcription assays, shows a ratio of 1:4 for region A to region B promoters. These data allowed us to predict the relative contributions of region A and B to mitochondrial transcript accumulation, based on a simple product of genome-form abundance and promoter strength. When promoter use was determined by using quantitative reverse transcriptase-PCR, however, we found that region A promoters were used at an unexpectedly high rate when upstream of cox2 and used less than expected when not upstream of cox2. Thus, the use of this set of promoters seems to respond to genomic context. These results suggest a role for intragenomic and intergenomic recombination in regulating plant mitochondrial gene expression.

Stoichiometric shifts in the common bean mitochondrial genome leading to male sterility and spontaneous reversion to fertility

The plant mitochondrial genome is characterized by a complex, multipartite structure. In cytoplasmic male-sterile (CMS) common bean, the sterility-inducing mitochondrial configuration maps as three autonomous DNA molecules, one containing the sterility-associated sequence pvs-or f 239. We constructed a physical map of the mitochondrial genome from the direct progenitors to the CMS cytoplasm and have shown that it maps as a single, circular master configuration. With long-exposure autoradiography of DNA gel blots and polymerase chain reaction analysis, we demonstrate that the three-molecule CMS-associated configuration was present at unusually low copy number within the progenitor genome and that the progenitor form was present substoichiometrically within the genome of the CMS line. Furthermore, upon spontaneous reversion to fertility, the progenitor genomic configuration as well as the molecule containing the pvs-or f 239 sterility-associated sequence were both maintained at substoichiometric levels within the revertant genome. In vitro mitochondrial incubation results demonstrated that the genomic shift of the pvs-or f 239-containing molecule to substoichiometric levels upon spontaneous reversion was a reversible phenomenon. Moreover, we demonstrate that substoichiometric forms, apparently silent with regard to gene expression, are transcriptionally and translationally active once amplified. Thus, copy number suppression may serve as an effective means of regulating gene expression in plant mitochondria.