Nad1 Intron Research Articles

PPR21 is involved in the splicing of nad2 introns via interacting with PPR–small MutS-related 1 and small PPR protein 2 and is essential to maize seed development

Pentatricopeptide repeat (PPR) proteins are a large group of eukaryote-specific RNA-binding proteins that play pivotal roles in plant organelle gene expression. Here, we report the function of PPR21 in mitochondrial intron splicing and its role in maize kernel development. PPR21 is a typical P-type PPR protein targeted to mitochondria. The ppr21 mutants are arrested in embryogenesis and endosperm development, leading to embryo lethality. Null mutations of PPR21 reduce the splicing efficiency of nad2 intron 1, 2, and 4 and impair the assembly and activity of mitochondrial complex I. Previous studies show that the P-type PPR protein EMP12 is required for the splicing of identical introns. However, our protein interaction analyses reveal that PPR21 does not interact with EMP12. Instead, both PPR21 and EMP12 interact with the small MutS-related (SMR) domain–containing PPR protein 1 (PPR-SMR1) and the short P-type PPR protein 2 (SPR2). PPR-SMR1 interacts with SPR2, and both proteins are required for the splicing of many introns in mitochondria, including nad2 intron 1, 2, and 4. These results suggest that a PPR21–(PPR-SMR1/SPR2)–EMP12 complex is involved in the splicing of nad2 introns in maize mitochondria.

PPR596 Is Required for nad2 Intron Splicing and Complex I Biogenesis in Arabidopsis.

Mitochondria are essential organelles that generate energy via oxidative phosphorylation. Plant mitochondrial genome encodes some of the respiratory complex subunits, and these transcripts require accurate processing, including C-to-U RNA editing and intron splicing. Pentatricopeptide repeats (PPR) proteins are involved in various organellar RNA processing events. PPR596, a P-type PPR protein, was previously identified to function in the C-to-U editing of mitochondrial rps3 transcripts in Arabidopsis. Here, we demonstrate that PPR596 functions in the cis-splicing of nad2 intron 3 in mitochondria. Loss of the PPR596 function affects the editing at rps3eU1344SS, impairs nad2 intron 3 splicing and reduces the mitochondrial complex I's assembly and activity, while inducing alternative oxidase (AOX) gene expression. This defect in nad2 intron splicing provides a plausible explanation for the slow growth of the ppr595 mutants. Although a few P-type PPR proteins are involved in RNA C-to-U editing, our results suggest that the primary function of PPR596 is intron splicing.

DEFECTIVE KERNEL 56 functions in mitochondrial RNA editing and maize seed development.

Proper seed development is essential for achieving grain production, successful seed germination, and seedling establishment in maize (Zea mays). In the past few decades, pentatricopeptide repeat (PPR) proteins have been proven to play an essential role in regulating the development of maize kernels through posttranscriptional RNA modification of mitochondrial genes. However, the underlying mechanisms remain largely unknown. Here, we characterized a mutant of DEFECTIVE KERNEL 56 (DEK56) with defective kernels that exhibited arrested development of both the embryo and endosperm. Accordingly, we isolated DEK56 through a map-based cloning strategy and found that it encoded an E subgroup PPR protein located in the mitochondria. Dysfunction of DEK56 resulted in altered cytidine (C)-to-uridine (U) editing efficiency at 48 editing sites across 21 mitochondrial transcripts. Notably, the editing efficiency of the maturase-related (matR)-1124 site was substantially reduced or abolished in the dek56 mutant. Furthermore, we found that the splicing efficiency of NADH dehydrogenase subunit 4 (nad4) Introns 1 and 3 was substantially reduced in dek56 kernels, which might be a consequence of the defective MatR function. Through a protein-protein interaction test, we hypothesized that DEK56 carries out its function by recruiting the PPR-DYW protein PPR motif, coiled-coil, and DYW domain-containing protein 1 (PCW1). This interaction is facilitated by Multiple Organellar RNA Editing Factors (ZmMORFs) and Glutamine-Rich Protein 23 (ZmGRP23). Based on these findings, we developed a working model of PPR-mediated mitochondrial processing that plays an essential role in the development of maize kernels. The present study will further broaden our understanding of PPR-mediated seed development and provide a theoretical basis for maize improvement.

The DEAD-box RNA helicase ZmRH48 is required for the splicing of multiple mitochondrial introns, mitochondrial complex biosynthesis, and seed development in maize.

RNA helicases participate in nearly all aspects of RNA metabolism by rearranging RNAs or RNA-protein complexes in an adenosine triphosphate-dependent manner. Due to the large RNA helicase families in plants, the precise roles of many RNA helicases in plant physiology and development remain to be clarified. Here, we show that mutations in maize (Zea mays) DEAD-box RNA helicase 48 (ZmRH48) impair the splicing of mitochondrial introns, mitochondrial complex biosynthesis, and seed development. Loss of ZmRH48 function severely arrested embryogenesis and endosperm development, leading to defective kernel formation. ZmRH48 is targeted to mitochondria, where its deficiency dramatically reduced the splicing efficiency of five cis-introns (nad5 intron 1; nad7 introns 1, 2, and 3; and ccmFc intron 1) and one trans-intron (nad2 intron 2), leading to lower levels of mitochondrial complexes I and III. ZmRH48 interacts with two unique pentatricopeptide repeat (PPR) proteins, PPR-SMR1 and SPR2, which are required for the splicing of over half of all mitochondrial introns. PPR-SMR1 interacts with SPR2, and both proteins interact with P-type PPR proteins and Zm-mCSF1 to facilitate intron splicing. These results suggest that ZmRH48 is likely a component of a splicing complex and is critical for mitochondrial complex biosynthesis and seed development.

Mito-nuclear discordance within Anthozoa, with notes on unique properties of their mitochondrial genomes

Whole mitochondrial genomes are often used in phylogenetic reconstruction. However, discordant patterns in species relationships between mitochondrial and nuclear phylogenies are commonly observed. Within Anthozoa (Phylum Cnidaria), mitochondrial (mt)-nuclear discordance has not yet been examined using a large and comparable dataset. Here, we used data obtained from target-capture enrichment sequencing to assemble and annotate mt genomes and reconstruct phylogenies for comparisons to phylogenies inferred from hundreds of nuclear loci obtained from the same samples. The datasets comprised 108 hexacorals and 94 octocorals representing all orders and > 50% of extant families. Results indicated rampant discordance between datasets at every taxonomic level. This discordance is not attributable to substitution saturation, but rather likely caused by introgressive hybridization and unique properties of mt genomes, including slow rates of evolution driven by strong purifying selection and substitution rate variation. Strong purifying selection across the mt genomes caution their use in analyses that rely on assumptions of neutrality. Furthermore, unique properties of the mt genomes were noted, including genome rearrangements and the presence of nad5 introns. Specifically, we note the presence of the homing endonuclease in ceriantharians. This large dataset of mitochondrial genomes further demonstrates the utility of off-target reads generated from target-capture data for mt genome assembly and adds to the growing knowledge of anthozoan evolution.

A mitochondrion-associated PPR protein, WBG1, regulates grain chalkiness in rice.

Rice kernel quality has vital commercial value. Grain chalkiness deteriorates rice's appearance and palatability. However, the molecular mechanisms that govern grain chalkiness remain unclear and may be regulated by many factors. In this study, we identified a stable hereditary mutant, white belly grain 1 (wbg1), which has a white belly in its mature grains. The grain filling rate of wbg1 was lower than that of the wild type across the whole filling period, and the starch granules in the chalky part were oval or round and loosely arranged. Map-based cloning showed that wbg1 was an allelic mutant of FLO10, which encodes a mitochondrion-targeted P-type pentatricopeptide repeat protein. Amino acid sequence analysis found that two PPR motifs present in the C-terminal of WBG1 were lost in wbg1. This deletion reduced the splicing efficiency of nad1 intron 1 to approximately 50% in wbg1, thereby partially reducing the activity of complex I and affecting ATP production in wbg1 grains. Furthermore, haplotype analysis showed that WBG1 was associated with grain width between indica and japonica rice varieties. These results suggested that WBG1 influences rice grain chalkiness and grain width by regulating the splicing efficiency of nad1 intron 1. This deepens understanding of the molecular mechanisms governing rice grain quality and provides theoretical support for molecular breeding to improve rice quality.

Rice FLOURY ENDOSPERM22, encoding a pentatricopeptide repeat protein, is involved in both mitochondrial RNA splicing and editing and is crucial for endosperm development.

Most of the reported P-type pentatricopeptide repeat (PPR) proteins play roles in organelle RNA stabilization and splicing. However, P-type PPRs involved in both RNA splicing and editing have rarely been reported, and their underlying mechanism remains largely unknown. Here, we report a rice floury endosperm22 (flo22) mutant with delayed amyloplast development in endosperm cells. Map-based cloning and complementation tests demonstrated that FLO22 encodes a mitochondrion-localized P-type PPR protein. Mutation of FLO22 resulting in defective trans-splicing of mitochondrial nad1 intron 1 and perhaps causing instability of mature transcripts affected assembly and activity of complex Ⅰ, and mitochondrial morphology and function. RNA-seq analysis showed that expression levels of many genes involved in starch and sucrose metabolism were significantly down-regulated in the flo22 mutant compared with the wild type, whereas genes related to oxidative phosphorylation and the tricarboxylic acid cycle were significantly up-regulated. In addition to involvement in splicing as a P-type PPR protein, we found that FLO22 interacted with DYW3, a DYW-type PPR protein, and they may function synergistically in mitochondrial RNA editing. The present work indicated that FLO22 plays an important role in endosperm development and plant growth by participating in nad1 maturation and multi-site editing of mitochondrial messager RNA.

ZmnMAT1, a nuclear-encoded type I maturase, is required for the splicing of mitochondrial Nad1 intron 1 and Nad4 intron 2.

Maturases can specifically bind to intron-containing pre-RNAs, folding them into catalytic structures that facilitate intron splicing in vivo. Plants possess four nuclear-encoded maturase-related factors (nMAT1-nMAT4) and some maturases have been shown to involve in the splicing of different mitochondrial group II introns; however, the specific biological functions of maturases in maize are largely uncharacterized. In this study, we identified a maize ZmnMAT1 gene, which encodes a mitochondrion-localized type I maturase with an RT domain at N-terminus and an X domain at C-terminus. Loss-of-function mutation in ZmnMAT1 significantly reduced the splicing efficiencies of Nad1 intron 1 and Nad4 intron 2, and showed arrested embryogenesis and endosperm development, which may be related to impaired mitochondrial ultrastructure and function due to the destruction of the assembly and activity of complex I. Direct physical interaction was undetectable between ZmnMAT1 and the proteins associated with the splicing of Nad1 intron 1 and/or Nad4 intron 2 by yeast two-hybrid assays, suggesting the complexity of group II intron splicing in plants.

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.

Maize PPR278 Functions in Mitochondrial RNA Splicing and Editing.

Pentatricopeptide repeat (PPR) proteins are a large protein family in higher plants and play important roles during seed development. Most reported PPR proteins function in mitochondria. However, some PPR proteins localize to more than one organelle; functional characterization of these proteins remains limited in maize (Zea mays L.). Here, we cloned and analyzed the function of a P-subfamily PPR protein, PPR278. Loss-function of PPR278 led to a lower germination rate and other defects at the seedling stage, as well as smaller kernels compared to the wild type. PPR278 was expressed in all investigated tissues. Furthermore, we determined that PPR278 is involved in the splicing of two mitochondrial transcripts (nad2 intron 4 and nad5 introns 1 and 4), as well as RNA editing of C-to-U sites in 10 mitochondrial transcripts. PPR278 localized to the nucleus, implying that it may function as a transcriptional regulator during seed development. Our data indicate that PPR278 is involved in maize seed development via intron splicing and RNA editing in mitochondria and has potential regulatory roles in the nucleus.

MISF2 Encodes an Essential Mitochondrial Splicing Cofactor Required for nad2 mRNA Processing and Embryo Development in Arabidopsis thaliana.

Mitochondria play key roles in cellular energy metabolism in eukaryotes. Mitochondria of most organisms contain their own genome and specific transcription and translation machineries. The expression of angiosperm mtDNA involves extensive RNA-processing steps, such as RNA trimming, editing, and the splicing of numerous group II-type introns. Pentatricopeptide repeat (PPR) proteins are key players in plant organelle gene expression and RNA metabolism. In the present analysis, we reveal the function of the MITOCHONDRIAL SPLICING FACTOR 2 gene (MISF2, AT3G22670) and show that it encodes a mitochondria-localized PPR protein that is crucial for early embryo development in Arabidopsis. Molecular characterization of embryo-rescued misf2 plantlets indicates that the splicing of nad2 intron 1, and thus respiratory complex I biogenesis, are strongly compromised. Moreover, the molecular function seems conserved between MISF2 protein in Arabidopsis and its orthologous gene (EMP10) in maize, suggesting that the ancestor of MISF2/EMP10 was recruited to function in nad2 processing before the monocot–dicot divergence ~200 million years ago. These data provide new insights into the function of nuclear-encoded factors in mitochondrial gene expression and respiratory chain biogenesis during plant embryo development.

Assembly and comparative analysis of the first complete mitochondrial genome of Acer truncatum Bunge: a woody oil-tree species producing nervonic acid

BackgroundAcer truncatum (purpleblow maple) is a woody tree species that produces seeds with high levels of valuable fatty acids (especially nervonic acid). The species is admired as a landscape plant with high developmental prospects and scientific research value. The A. truncatum chloroplast genome has recently been reported; however, the mitochondrial genome (mitogenome) is still unexplored.ResultsWe characterized the A. truncatum mitogenome, which was assembled using reads from PacBio and Illumina sequencing platforms, performed a comparative analysis against different species of Acer. The circular mitogenome of A. truncatum has a length of 791,052 bp, with a base composition of 27.11% A, 27.21% T, 22.79% G, and 22.89% C. The A. truncatum mitogenome contains 62 genes, including 35 protein-coding genes, 23 tRNA genes and 4 rRNA genes. We also examined codon usage, sequence repeats, RNA editing and selective pressure in the A. truncatum mitogenome. To determine the evolutionary and taxonomic status of A. truncatum, we conducted a phylogenetic analysis based on the mitogenomes of A. truncatum and 25 other taxa. In addition, the gene migration from chloroplast and nuclear genomes to the mitogenome were analyzed. Finally, we developed a novel NAD1 intron indel marker for distinguishing several Acer species.ConclusionsIn this study, we assembled and annotated the mitogenome of A. truncatum, a woody oil-tree species producing nervonic acid. The results of our analyses provide comprehensive information on the A. truncatum mitogenome, which would facilitate evolutionary research and molecular barcoding in Acer.

CFM6 is an Essential CRM Protein Required for the Splicing of nad5 Transcript in Arabidopsis Mitochondria.

Plant chloroplast RNA splicing and ribosome maturation (CRM)-domain-containing proteins are capable of binding RNA to facilitate the splicing of group I or II introns in chloroplasts, but their functions in mitochondria are less clear. In the present study, Arabidopsis thaliana CFM6, a protein with a single CRM domain, was expressed in most plant tissues, particularly in flower tissues, and restricted to mitochondria. Mutation of CFM6 causes severe growth defects, including stunted growth, curled leaves, delayed embryogenesis and pollen development. CFM6 functions specifically in the splicing of group II intron 4 of nad5, which encodes a subunit of mitochondrial complex I, as evidenced by the loss of nad5 intron 4 splicing and high accumulation of its pretranscripts in cfm6 mutants. The phenotypic and splicing defects of cfm6 were rescued in transgenic plants overexpressing 35S::CFM6-YFP. Splicing failure in cfm6 also led to the loss of complex I activity and to its improper assembly. Moreover, dysfunction of complex I induced the expression of proteins or genes involved in alternative respiratory pathways in cfm6. Collectively, CFM6, a previously uncharacterized CRM domain-containing protein, is specifically involved in the cis-splicing of nad5 intron 4 and plays a pivotal role in mitochondrial complex I biogenesis and normal plant growth.

OsNBL3, a mitochondrion-localized pentatricopeptide repeat protein, is involved in splicing nad5 intron 4 and its disruption causes lesion mimic phenotype with enhanced resistance to biotic and abiotic stresses.

SummaryLesion mimic mutants are used to elucidate mechanisms controlling plant responses to pathogen attacks and environmental stresses. Although dozens of genes had been functionally demonstrated to be involved in lesion mimic phenotype in several plant species, the molecular mechanisms underlying the hypersensitive response are largely unknown. Here, a rice (Oryza sativa) lesion mimic mutant natural blight leaf 3 (nbl3) was identified from T‐DNA insertion lines. The causative gene, OsNBL3, encodes a mitochondrion‐localized pentatricopeptide repeat (PPR) protein. The nbl3 mutant exhibited spontaneous cell death response and H2O2 accumulation, and displayed enhanced resistance to the fungal and bacterial pathogens Magnaporthe oryzae and Xanthomonas oryzae pv. oryzae. This resistance was consistent with the up‐regulation of several defence‐related genes; thus, defence responses were induced in nbl3. RNA interference lines of OsNBL3 exhibited enhanced disease resistance similar to that of nbl3, while the disease resistance in overexpression lines did not differ from that of the wild type. In addition, nbl3 displayed improved tolerance to salt, accompanied by up‐regulation of several salt‐associated marker genes. OsNBL3 was found to mainly participate in the splicing of mitochondrial gene nad5 intron 4. Disruption of OsNBL3 leads to the reduction in complex I activity, the elevation of alternative respiratory pathways and the destruction of mitochondrial morphology. Overall, the results demonstrated that the PPR protein‐coding gene OsNBL3 is essential for mitochondrial development and functions, and its disruption causes the lesion mimic phenotype and enhances disease resistance and tolerance to salt in rice.

NMAT3 is an essential maturase splicing factor required for holo-complexI biogenesis and embryo development in Arabidopsis thaliana plants.

Group-II introns are self-splicing mobile genetic elements consisting of catalytic intron-RNA and its related intron-encoded splicing maturase protein cofactor. Group-II sequences are particularly plentiful within the mitochondria of land plants, where they reside within many critical gene loci. During evolution, the plant organellar introns have degenerated, such as they lack regions that are are required for splicing, and also lost their evolutionary related maturase proteins. Instead, for their splicing the organellar introns in plants rely on different host-acting protein cofactors, which may also provide a means to link cellular signals with respiratory functions. The nuclear genome of Arabidopsis thaliana encodes four maturase-related factors. Previously, we showed that three of the maturases, nMAT1, nMAT2 and nMAT4, function in the excision of different group-II introns in Arabidopsis mitochondria. The function of nMAT3 (encoded by the At5g04050 gene locus) was found to be essential during early embryogenesis. Using a modified embryo-rescue method, we show that nMAT3-knockout plants are strongly affected in the splicing of nad1 introns1, 3 and 4 in Arabidopsis mitochondria, resulting in complex-I biogenesis defects and altered respiratory activities. Functional complementation of nMAT3 restored the organellar defects and embryo-arrested phenotypes associated with the nmat3 mutant line. Notably, nMAT3 and nMA4 were found to act on the same RNA targets but have no redundant functions in the splicing of nad1 transcripts. The two maturases, nMAT3 and nMAT4 are likely to cooperate together in the maturation of nad1 pre-RNAs. Our results provide important insights into the roles of maturases in mitochondria gene expression and the biogenesis of the respiratory system during early plant life.

Rice FLOURY SHRUNKEN ENDOSPERM 5 Encodes a Putative Plant Organelle RNA Recognition Protein that Is Required for cis-Splicing of Mitochondrial nad4 Intron 1

BackgroundThe sequences of several important mitochondrion-encoded genes involved in respiration in higher plants are interrupted by introns. Many nuclear-encoded factors are involved in splicing these introns, but the mechanisms underlying this splicing remain unknown.ResultsWe isolated and characterized a rice mutant named floury shrunken endosperm 5 (fse5). In addition to having floury shrunken endosperm, the fse5 seeds either failed to germinate or produced seedlings which grew slowly and died ultimately. Fse5 encodes a putative plant organelle RNA recognition (PORR) protein targeted to mitochondria. Mutation of Fse5 hindered the splicing of the first intron of nad4, which encodes an essential subunit of mitochondrial NADH dehydrogenase complex I. The assembly and NADH dehydrogenase activity of complex I were subsequently disrupted by this mutation, and the structure of the mitochondria was abnormal in the fse5 mutant. The FSE5 protein was shown to interact with mitochondrial intron splicing factor 68 (MISF68), which is also a splicing factor for nad4 intron 1 identified previously via yeast two-hybrid (Y2H) assays.ConclusionFse5 which encodes a PORR domain-containing protein, is essential for the splicing of nad4 intron 1, and loss of Fse5 function affects seed development and seedling growth.

Nuclear-Encoded Maturase Protein 3 Is Required for the Splicing of Various Group II Introns in Mitochondria during Maize (Zea mays L.) Seed Development.

Splicing of plant organellar group II introns from precursor-RNA transcripts requires the assistance of nuclear-encoded splicing factors. Maturase (nMAT) is one such factor, as its three homologs (nMAT1, 2 and 4) have been identified as being required for the splicing of various mitochondrial introns in Arabidopsis. However, the function of nMAT in maize (Zea mays L.) is unknown. In this study, we identified a seed development mutant, empty pericarp 2441 (emp2441) from maize, which showed severely arrested embryogenesis and endosperm development. Positional cloning and transgenic complementation assays revealed that Emp2441 encodes a maturase-related protein, ZmnMAT3. ZmnMAT3 is highly expressed during seed development and its protein locates to the mitochondria. The loss of function of ZmnMAT3 resulted in the reduced splicing efficiency of various mitochondrial group II introns, particularly of the trans-splicing of nad1 introns 1, 3 and 4, which consequently abolished the transcript of nad1 and severely impaired the assembly and activity of mitochondrial complex I. Moreover, the Zmnmat3 mutant showed defective mitochondrial structure and exhibited expression and activity of alternative oxidases. These results indicate that ZmnMAT3 is essential for mitochondrial complex I assembly during kernel development in maize.

OsPPR939, a nad5 splicing factor, is essential for plant growth and pollen development in rice

Key messageP-subfamily PPR protein OsPPR939, which can be phosphorylated by OsS6K1, regulates plant growth and pollen development by involving in the splicing of mitochondrial nad5 introns 1, 2, and 3.In land plants, pentatricopeptide repeat (PPR) proteins play key roles in mitochondrial group II intron splicing, but how these nucleus-encoded proteins are imported into mitochondria is unknown. To date, a few PPR proteins have been characterized in rice (Oryza sativa). Here, we demonstrate that the mitochondrion-localized P-subfamily PPR protein OsPPR939 is required for the splicing of nad5 introns 1, 2, and 3 in rice. Complete knockout or partial disruption of OsPPR939 function resulted in different degrees of growth retardation and pollen sterility. The dramatically reduced splicing efficiency of these introns in osppr939-4 and osppr939-5 led to reduced mitochondrial complex I abundance and activity and enhanced expression of alternative respiratory pathway genes. Complementation with OsPPR939 rescued the defective plant morphology of osppr939-4 and restored its decreased splicing efficiency of nad5 introns 1, 2, and 3. Therefore, OsPPR939 plays crucial roles in plant growth and pollen development by splicing mitochondrial nad5 introns 1, 2, and 3. More importantly, the 12th amino acid Ser in the N-terminal targeting sequence of OsPPR939 is phosphorylated by OsS6K1, and truncated OsPPR939 with a non-phosphorylatable S12A mutation in its presequence could not be imported into mitochondria, suggesting that phosphorylation of this amino acid plays an important role in the mitochondrial import of OsPPR939. To our knowledge, the 12th residue Ser on OsPPR939 is the first experimentally proven phosphorylation site in PPR proteins. Our results provide a basis for investigating the regulatory mechanism of PPR proteins at the post-translational level.

Empty Pericarp24 and Empty Pericarp25 Are Required for the Splicing of Mitochondrial Introns, Complex I Assembly, and Seed Development in Maize

RNA splicing is an essential post-transcriptional regulation in plant mitochondria and chloroplasts. As the mechanism of RNA splicing remains obscure, identification and functional elucidation of new splicing factors are necessary. Through a characterization of two maize mutants, we cloned Empty pericarp 24 (Emp24) and Empty pericarp 25 (Emp25). Both Emp24 and Emp25 encode mitochondrion-targeted P-type PPR proteins. EMP24 is required for the splicing of nad4 introns 1 and 3, which was reported (Ren Z. et al., 2019), and EMP25 functions in the splicing of nad5 introns 1, 2, and 3. Absence of either Nad4 or Nad5 proteins blocks the assembly of mitochondrial complex I, resulting in the formation of a sub-sized complex I of similar size in both mutants. Mass spectrometry identification revealed that the subcomplexes in both mutants lack an identical set of proteins of complex I. These results indicate that EMP24 and EMP25 function in the splicing of nad4 and nad5 introns, respectively, and are essential to maize kernel development. The identification of the subcomplexes provides genetic and molecular insights into the modular complex I assembly pathway in maize.

Differential RNA Editing and Intron Splicing in Soybean Mitochondria during Nodulation.

Nitrogen fixation in soybean consumes a tremendous amount of energy, leading to substantial differences in energy metabolism and mitochondrial activities between nodules and uninoculated roots. While C-to-U RNA editing and intron splicing of mitochondrial transcripts are common in plant species, their roles in relation to nodule functions are still elusive. In this study, we performed RNA-seq to compare transcript profiles and RNA editing of mitochondrial genes in soybean nodules and roots. A total of 631 RNA editing sites were identified on mitochondrial transcripts, with 12% or 74 sites differentially edited among the transcripts isolated from nodules, stripped roots, and uninoculated roots. Eight out of these 74 differentially edited sites are located on the matR transcript, of which the degrees of RNA editing were the highest in the nodule sample. The degree of mitochondrial intron splicing was also examined. The splicing efficiencies of several introns in nodules and stripped roots were higher than in uninoculated roots. These include nad1 introns 2/3/4, nad4 intron 3, nad5 introns 2/3, cox2 intron 1, and ccmFc intron 1. A greater splicing efficiency of nad4 intron 1, a higher NAD4 protein abundance, and a reduction in supercomplex I + III2 were also observed in nodules, although the causal relationship between these observations requires further investigation.