Root Primordium Defective 1 Encodes an Essential PORR Protein Required for the Splicing of Mitochondria Encoded Group II Introns and for Respiratory Complex I Biogenesis.

Cellular respiration involves complex organellar metabolic activities that are pivotal for plant growth and development. Mitochondria contain their own genetic system (mitogenome, mtDNA), which encodes key elements of the respiratory machinery. Plant mtDNAs are notably larger than their counterparts in Animalia, with complex genome organization and gene-expression characteristics. The maturation of the plant mitochondrial transcripts involves extensive RNA editing, trimming and splicing events. These essential processing steps rely on the activities of numerous nuclear-encoded cofactors, which may also play key regulatory roles in mitochondrial biogenesis and function, and hence in plant physiology. Proteins that harbor the Plant Organelle RNA Recognition (PORR) domain are represented in a small gene family in plants. Several PORR members, including WTF1, WTF9 and LEFKOTHEA, are known to act in the splicing of organellar group II introns in angiosperms. The AT4G33495 gene-locus encodes an essential PORR-protein in Arabidopsis, termed as ROOT PRIMORDIUM DEFECTIVE 1 (RPD1). A null mutation of At.RPD1 causes arrest in early embryogenesis, while the missense mutant lines, rpd1.1 and rpd1.2, exhibit a strong impairment in root development and retarded growth phenotypes, especially under high-temperature conditions. Here, we further show that RPD1 functions in the splicing of introns that reside in the coding regions of various complex I (CI) subunits (i.e., nad2, nad4, nad5 and nad7), as well as in the maturation of the ribosomal rps3 pre-RNA in Arabidopsis mitochondria. The altered growth and developmental phenotypes and modified respiration activities are tightly correlated with respiratory chain CI defects in rpd1 mutants.

Topologies of N6 -adenosine methylation (m6 A) in land plant mitochondria and their putative effects on organellar gene expression.

Mitochondria serve as major sites of ATP production and play key roles in many other metabolic processes that are critical to the cell. As relicts of an ancient bacterial endosymbiont, mitochondria contain their own hereditary material (i.e. mtDNA, or mitogenome) and a machinery for protein biosynthesis. The expression of the mtDNA in plants is complex, particularly at the post-transcriptional level. Following transcription, the polycistronic pre-RNAs undergo extensive modifications, including trimming, splicing and editing, before being translated by organellar ribosomes. Our study focuses on N6 -methylation of adenosine ribonucleotides (m6 A-RNA) in plant mitochondria. m6 A is a prevalent modification in nuclear-encoded mRNAs. The biological significance of this dynamic modification is under investigation, but it is widely accepted that m6 A mediates structural switches that affect RNA stability and/or activity. Using m6 A-pulldown/RNA-seq (m6 A-RIP-seq) assays of Arabidopsis and cauliflower mitochondria, we provide information on the m6 A-RNA landscapes in Arabidopsis thaliana and Brassica oleracea mitochondria. The results show that m6 A targets different types of mitochondrial transcripts, including known genes, mtORFs, as well as non-coding (transcribed intergenic) RNA species. While ncRNAs undergo multiple m6 A modifications, N6 -methylation of adenosine residues with mRNAs seem preferably positioned near start codons and may modulate their translatability.

Correction: Control of organelle gene expression by the mitochondrial transcription termination factor mTERF22 in Arabidopsis thaliana plants.

[This corrects the article DOI: 10.1371/journal.pone.0201631.].

Control of organelle gene expression by the mitochondrial transcription termination factor mTERF22 in Arabidopsis thaliana plants.

Mitochondria are key sites for cellular energy metabolism and are essential to cell survival. As descendants of eubacterial symbionts (specifically α-proteobacteria), mitochondria contain their own genomes (mtDNAs), RNAs and ribosomes. Plants need to coordinate their energy demands during particular growth and developmental stages. The regulation of mtDNA expression is critical for controlling the oxidative phosphorylation capacity in response to physiological or environmental signals. The mitochondrial transcription termination factor (mTERF) family has recently emerged as a central player in mitochondrial gene expression in various eukaryotes. Interestingly, the number of mTERFs has been greatly expanded in the nuclear genomes of plants, with more than 30 members in different angiosperms. The majority of the annotated mTERFs in plants are predicted to be plastid- or mitochondria-localized. These are therefore expected to play important roles in organellar gene expression in angiosperms. Yet, functions have been assigned to only a small fraction of these factors in plants. Here, we report the characterization of mTERF22 (At5g64950) which functions in the regulation of mtDNA transcription in Arabidopsis thaliana. GFP localization assays indicate that mTERF22 resides within the mitochondria. Disruption of mTERF22 function results in reduced mtRNA accumulation and altered organelle biogenesis. Transcriptomic and run-on experiments suggest that the phenotypes of mterf22 mutants are attributable, at least in part, to altered mitochondria transcription, and indicate that mTERF22 affects the expression of numerous mitochondrial genes in Arabidopsis plants.

Resequencing of a mutant bearing an iron starvation recovery phenotype defines Slr1658 as a new player in the regulatory network of a model cyanobacterium.

Photosynthetic microorganisms encounter an erratic nutrient environment characterized by periods of iron limitation and sufficiency. Surviving in such an environment requires mechanisms for handling these transitions. Our study identified a regulatory system involved in the process of recovery from iron limitation in cyanobacteria. We set out to study the role of bacterioferritin co-migratory proteins during transitions in iron bioavailability in the cyanobacterium Synechocystis sp. PCC 6803 using knockout strains coupled with physiological and biochemical measurements. One of the mutants displayed slow recovery from iron limitation. However, we discovered that the cause of the phenotype was not the intended knockout but rather the serendipitous selection of a mutation in an unrelated locus, slr1658. Bioinformatics analysis suggested similarities to two-component systems and a possible regulatory role. Transcriptomic analysis of the recovery from iron limitation showed that the slr1658 mutation had an extensive effect on the expression of genes encoding regulatory proteins, proteins involved in the remodeling and degradation of the photosynthetic apparatus and proteins modulating electron transport. Most significantly, expression of the cyanobacterial homologue of the cyclic electron transport protein PGR5 was upregulated 1000-fold in slr1658 disruption mutants. pgr5 transcripts in the Δslr1658 mutant retained these high levels under a range of stress and recovery conditions. The results suggest that slr1658 is part of a regulatory operon that, among other aspects, affects the regulation of alternative electron flow. Disruption of its function has deleterious results under oxidative stress promoting conditions.

Analysis of the Roles of the Arabidopsis nMAT2 and PMH2 Proteins Provided with New Insights into the Regulation of Group II Intron Splicing in Land-Plant Mitochondria.

Plant mitochondria are remarkable with respect to the presence of numerous group II introns which reside in many essential genes. The removal of the organellar introns from the coding genes they interrupt is essential for respiratory functions, and is facilitated by different enzymes that belong to a diverse set of protein families. These include maturases and RNA helicases related proteins that function in group II intron splicing in different organisms. Previous studies indicate a role for the nMAT2 maturase and the RNA helicase PMH2 in the maturation of different pre-RNAs in Arabidopsis mitochondria. However, the specific roles of these proteins in the splicing activity still need to be resolved. Using transcriptome analyses of Arabidopsis mitochondria, we show that nMAT2 and PMH2 function in the splicing of similar subsets of group II introns. Fractionation of native organellar extracts and pulldown experiments indicate that nMAT2 and PMH2 are associated together with their intron-RNA targets in large ribonucleoprotein particle in vivo. Moreover, the splicing efficiencies of the joint intron targets of nMAT2 and PMH2 are more strongly affected in a double nmat2/pmh2 mutant-line. These results are significant as they may imply that these proteins serve as components of a proto-spliceosomal complex in plant mitochondria.

The Reverse Transcriptase/RNA Maturase Protein MatR Is Required for the Splicing of Various Group II Introns in Brassicaceae Mitochondria.

Group II introns are large catalytic RNAs that are ancestrally related to nuclear spliceosomal introns. Sequences corresponding to group II RNAs are found in many prokaryotes and are particularly prevalent within plants organellar genomes. Proteins encoded within the introns themselves (maturases) facilitate the splicing of their own host pre-RNAs. Mitochondrial introns in plants have diverged considerably in sequence and have lost their maturases. In angiosperms, only a single maturase has been retained in the mitochondrial DNA: the matR gene found within NADH dehydrogenase 1 (nad1) intron 4. Its conservation across land plants and RNA editing events, which restore conserved amino acids, indicates that matR encodes a functional protein. However, the biological role of MatR remains unclear. Here, we performed an in vivo investigation of the roles of MatR in Brassicaceae. Directed knockdown of matR expression via synthetically designed ribozymes altered the processing of various introns, including nad1 i4. Pull-down experiments further indicated that MatR is associated with nad1 i4 and several other intron-containing pre-mRNAs. MatR may thus represent an intermediate link in the gradual evolutionary transition from the intron-specific maturases in bacteria into their versatile spliceosomal descendants in the nucleus. The similarity between maturases and the core spliceosomal Prp8 protein further supports this intriguing theory.

Organellar maturases: A window into the evolution of the spliceosome.

During the evolution of eukaryotic genomes, many genes have been interrupted by intervening sequences (introns) that must be removed post-transcriptionally from RNA precursors to form mRNAs ready for translation. The origin of nuclear introns is still under debate, but one hypothesis is that the spliceosome and the intron-exon structure of genes have evolved from bacterial-type group II introns that invaded the eukaryotic genomes. The group II introns were most likely introduced into the eukaryotic genome from an α-proteobacterial predecessor of mitochondria early during the endosymbiosis event. These self-splicing and mobile introns spread through the eukaryotic genome and later degenerated. Pieces of introns became part of the general splicing machinery we know today as the spliceosome. In addition, group II introns likely brought intron maturases with them to the nucleus. Maturases are found in most bacterial introns, where they act as highly specific splicing factors for group II introns. In the spliceosome, the core protein Prp8 shows homology to group II intron-encoded maturases. While maturases are entirely intron specific, their descendant of the spliceosomal machinery, the Prp8 protein, is an extremely versatile splicing factor with multiple interacting proteins and RNAs. How could such a general player in spliceosomal splicing evolve from the monospecific bacterial maturases? Analysis of the organellar splicing machinery in plants may give clues on the evolution of nuclear splicing. Plants encode various proteins which are closely related to bacterial maturases. The organellar genomes contain one maturase each, named MatK in chloroplasts and MatR in mitochondria. In addition, several maturase genes have been found in the nucleus as well, which are acting on mitochondrial pre-RNAs. All plant maturases show sequence deviation from their progenitor bacterial maturases, and interestingly are all acting on multiple organellar group II intron targets. Moreover, they seem to function in the splicing of group II introns together with a number of additional nuclear-encoded splicing factors, possibly acting as an organellar proto-spliceosome. Together, this makes them interesting models for the early evolution of nuclear spliceosomal splicing. In this review, we summarize recent advances in our understanding of the role of plant maturases and their accessory factors in plants. This article is part of a Special Issue entitled: Chloroplast Biogenesis.