In addition to the nucleus, mitochondria and chloroplasts in plant cells also contain genomes. Efficient DNA repair pathways are crucial in these organelles to fix damage resulting from endogenous ...and exogenous factors. Plant organellar genomes are complex compared with their animal counterparts, and although several plant-specific mediators of organelle DNA repair have been reported, many regulators remain to be identified. Here, we show that a mitochondrial SWI/SNF (nucleosome remodeling) complex B protein, SWIB5, is capable of associating with mitochondrial DNA (mtDNA) in Arabidopsis thaliana. Gain- and loss-of-function mutants provided evidence for a role of SWIB5 in influencing mtDNA architecture and homologous recombination at specific intermediate-sized repeats both under normal and genotoxic conditions. SWIB5 interacts with other mitochondrial SWIB proteins. Gene expression and mutant phenotypic analysis of SWIB5 and SWIB family members suggests a link between organellar genome maintenance and cell proliferation. Taken together, our work presents a protein family that influences mtDNA architecture and homologous recombination in plants and suggests a link between organelle functioning and plant development.
In
there are more than 600 C-to-U RNA editing events in the mitochondria and at least 44 in the chloroplasts. Pentatricopeptide repeat (PPR) proteins provide the specificity for these reactions. They ...recognize RNA sequences in a partially predictable fashion via key amino acids at the fifth and last position in each PPR motif that bind to individual ribonucleotides. A combined approach of RNA-Seq, mutant complementation, electrophoresis of mitochondrial protein complexes and Western blotting allowed us to show that MEF100, a PPR protein identified in a genetic screen for mutants resistant to an inhibitor of γ -glutamylcysteine synthetase, is required for the editing of
-493,
-403,
-698 and
-356 sites in Arabidopsis mitochondria. The absence of editing in
leads to a decrease in mitochondrial Complex I activity, which probably explains the physiological phenotype. Some plants have lost the requirement for MEF100 at one or more of these sites through mutations in the mitochondrial genome. We show that loss of the requirement for MEF100 editing leads to divergence in the MEF100 binding site.
Pentatricopeptide repeat (PPR) proteins are considered a potential tool for manipulating organelle gene expression in plants because they can recognise a wide range of different RNA sequences, and ...the molecular basis for this sequence recognition is partially known and understood. A library of redesigned PPR proteins related to restorer-of-fertility proteins was created and transformed into plants in order to target mitochondrial transcripts. Ninety different variants tested in vivo showed a wide range of phenotypes. One of these lines, which displayed slow growth and downward curled leaves, showed a clear reduction in complex V. The phenotype was due to a specific cleavage of atp1 transcripts induced by a modified PPR protein from the library, validating the use of this library as a source of mitochondrial ‘mutants’. This study is a step towards developing specific RNA targeting tools using PPR proteins that can be aimed at desired targets.
The molecular chaperone heat shock protein101 (HSP101) is required for acquired thermotolerance in plants and other organisms. To identify factors that interact with HSP101 or that are involved in ...thermotolerance, we screened for extragenic suppressors of a dominant-negative alíele of Arabidopsis thaliana HSP101, hot1-4. One suppressor, shoti (for suppressor of hot1-4 1), encodes a mitochondrial transcription termination factor (mTERF)-related protein, one of 35 Arabidopsis mTERFs about which there is limited functional data. Missense (shoti-1) and T-DNA insertion (shoti-2) mutants suppress the hot1-4 heat-hypersensitive phenotype. Furthermore, shot1-2 suppresses other heat-sensitive mutants, and shot1-2 alone is more heat tolerant than the wild type. SHOT1 resides in mitochondria, indicating it functions independently of cytosolic/nuclear HSP101. Microarray analysis suggests altered mitochondrial function and/or retrograde signaling in shoti-2 increases transcripts of other HSPs and alters expression of redox-related genes. Reduced oxidative damage is the likely cause of shoti thermotolerance, indicating HSP101 repairs protein oxidative damage and/or reduced oxidative damage allows recovery in the absence of HSP101. Changes in organelle-encoded transcripts in shoti demonstrate that SHOT1 is involved in organelle gene regulation. The heat tolerance of shoti emphasizes the importance of mitochondria in stress tolerance, and defining its function may provide insights into control of oxidative damage for engineering stress-resistant plants.
In higher plants, formate dehydrogenase (FDH, EC1.2.1.2.) catalyzes the NAD-linked oxidation of formate to CO₂, and FDH transcript accumulation has been reported after various abiotic stresses. By ...sequencing a Phaseolus vulgaris BAC clone encompassing a CC-NBS-LRR gene rich region of the B4 resistance gene cluster, we identified three FDH-encoding genes. FDH is present as a single copy gene in the Arabidopsis thaliana genome, and public database searches confirm that FDH is a low copy gene in plant genomes, since only 33 FDH homologs were identified from 27 plant species. Three independent prediction programs (Predotar, TargetP and Mitoprot) used on this large subset of 33 plant FDHs, revealed that mitochondrial localization of FDH might be the rule in higher plants. A phylogenetic analysis suggests a scenario of local FDH gene duplication in an ancestor of the Phaseoleae followed by another more recent duplication event after bean/soybean divergence. The expression levels of two common bean FDH genes under different treatments were investigated by quantitative RT-PCR analysis. FDH genes are differentially up-regulated after biotic and abiotic stresses (infection with the fungus Colletotrichum lindemuthianum, and dark treatment, respectively). The present study provides the first report of FDH transcript accumulation after biotic stress, suggesting the involvement of FDH in the pathogen resistance process.
Summary
Angiosperm mitochondria encode approximately 20 group II introns, which interrupt genes involved in the biogenesis and function of the respiratory chain. Nucleus‐encoded splicing factors have ...been identified for approximately half of these introns. The splicing factors derive from several protein families defined by atypical RNA binding domains that function primarily in organelles. We show here that the Arabidopsis protein WTF9 is essential for the splicing of group II introns in two mitochondrial genes for which splicing factors had not previously been identified: rpl2 and ccmFC. WTF9 harbors a recently recognized RNA binding domain, the PORR domain, which was originally characterized in the chloroplast splicing factor WTF1. These findings show that the PORR domain family also functions in plant mitochondria, and highlight the parallels between the machineries for group II intron splicing in plant mitochondria and chloroplasts. In addition, we used the splicing defects in wtf9 mutants as a means to functionally characterize the mitochondrial rpl2 and ccmFC genes. Loss of ccmFC expression correlates with the loss of cytochromes c and c1, confirming a role for ccmFC in cytochrome biogenesis. By contrast, our results strongly suggest that splicing is not essential for the function of the mitochondrial rpl2 gene, and imply that the Rpl2 fragment encoded by rpl2 exon 1 functions in concert with a nuclear gene product that provides the remainder of this essential ribosomal protein in trans.
Spontaneous mutations are rare in mitochondria and the lack of mitochondrial transformation methods has hindered genetic analyses. We show that a custom-designed RNA-binding pentatricopeptide repeat ...(PPR) protein binds and specifically induces cleavage of ATP synthase subunit1 (atp1) mRNA in mitochondria, significantly decreasing the abundance of the Atp1 protein and the assembled F1Fo ATP synthase in Arabidopsis (Arabidopsis thaliana). The transformed plants are characterized by delayed vegetative growth and reduced fertility. Five-fold depletion of Atp1 level was accompanied by a decrease in abundance of other ATP synthase subunits and lowered ATP synthesis rate of isolated mitochondria, but no change to mitochondrial electron transport chain complexes, adenylates, or energy charge in planta. Transcripts for amino acid transport and a variety of stress response processes were differentially expressed in lines containing the PPR protein, indicating changes to achieve cellular homeostasis when ATP synthase was highly depleted. Leaves of ATP synthase-depleted lines showed higher respiratory rates and elevated steady-state levels of numerous amino acids, most notably of the serine family. The results show the value of using custom-designed PPR proteins to influence the expression of specific mitochondrial transcripts to carry out reverse genetic studies on mitochondrial gene functions and the consequences of ATP synthase depletion on cellular functions in Arabidopsis.
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.
We report the partial complementation and subsequent comparative molecular analysis of two nonviable mutants impaired in chloroplast translation, one (emb2394) lacking the RPL6 protein, and the other ...(emb2654) carrying a mutation in a gene encoding a P-class pentatricopeptide repeat protein. We show that EMB2654 is required for the trans-splicing of the plastid rps12 transcript and that therefore the emb2654 mutant lacks Rps12 protein and fails to assemble the small subunit of the plastid ribosome, explaining the loss of plastid translation and consequent embryo-lethal phenotype. Predictions of the EMB2654 binding site match a small RNA "footprint" located on the 5ʹ half of the trans-spliced intron that is almost absent in the partially complemented mutant. EMB2654 binds sequence specifically to this target sequence in vitro. Altered patterns in nuclease-protected small RNA fragments in emb2654 show that EMB2654 binding must be an early step in, or prior to, the formation of a large protein-RNA complex covering the free ends of the two rps12 intron halves.
Summary
Angiosperm mitochondria encode approximately 20 group II introns, which interrupt genes involved in the biogenesis and function of the respiratory chain. Nucleus‐encoded splicing factors have ...been identified for approximately half of these introns. The splicing factors derive from several protein families defined by atypical RNA binding domains that function primarily in organelles. We show here that the Arabidopsis protein WTF9 is essential for the splicing of group II introns in two mitochondrial genes for which splicing factors had not previously been identified:
rpl2
and
ccmF
C
. WTF9 harbors a recently recognized RNA binding domain, the PORR domain, which was originally characterized in the chloroplast splicing factor WTF1. These findings show that the PORR domain family also functions in plant mitochondria, and highlight the parallels between the machineries for group II intron splicing in plant mitochondria and chloroplasts. In addition, we used the splicing defects in
wtf9
mutants as a means to functionally characterize the mitochondrial
rpl2
and
ccmF
C
genes. Loss of
ccmF
C
expression correlates with the loss of cytochromes
c
and
c
1
, confirming a role for
ccmF
C
in cytochrome biogenesis. By contrast, our results strongly suggest that splicing is not essential for the function of the mitochondrial
rpl2
gene, and imply that the Rpl2 fragment encoded by
rpl2
exon 1 functions in concert with a nuclear gene product that provides the remainder of this essential ribosomal protein
in trans.
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