Defective kernel 58 encodes an Rrp15p domain-containing protein essential to ribosome biogenesis and seed development in maize.

Ribosome biogenesis is a highly dynamic and orchestrated process facilitated by hundreds of ribosomal biogenesis factors and small nucleolar RNAs. While many of the advances are derived from studies in yeast, ribosome biogenesis remains largely unknown in plants despite its importance to plant growth and development. Through characterizing the maize (Zea mays) defective kernel and embryo-lethal mutant dek58, we show that DEK58 encodes an Rrp15p domain-containing protein with 15.3% identity to yeast Rrp15. Over-expression of DEK58 rescues the mutant phenotype. DEK58 is localized in the nucleolus. Ribosome profiling and RNA gel blot analyses show that the absence of DEK58 reduces ribosome assembly and impedes pre-rRNA processing, accompanied by the accumulation of nearly all the pre-rRNA processing intermediates and the production of an aberrant processing product P-25S*. DEK58 interacts with ZmSSF1, a maize homolog of the yeast Ssf1 in the 60S processome. DEK58 and ZmSSF1 interact with ZmCK2α, a putative component of the yeast UTP-C complex involved in the small ribosomal subunit processome. These results demonstrate that DEK58 is essential to seed development in maize. It functions in the early stage of pre-rRNA processing in ribosome biogenesis, possibly through interacting with ZmSSF1 and ZmCK2α in maize.

Homologous recombination deficiency status predicts response to platinum-based chemotherapy in Chinese patients with high-grade serous ovarian carcinoma.

BACKGROUND: Homologous Recombination Deficiency (HRD) is a predictive biomarker for ovarian cancer treated with PARP inhibitors or for breast cancer treated with first-line platinum-based chemotherapy. However, limited research is documented on platinum-based treatment prediction with HRD as a biomarker in ovarian cancer patients, especially in the Chinese population. METHODS: We investigated the association between HRD status and the response of platinum-based chemotherapy in 240 Chinese HGSOC patients. RESULTS: The Pt-sensitive patients showed higher HRD scores than Pt-resistant ones, but this was not significant(median: 42.6 vs. 31.6, p = 0.086). (Pt)-sensitive rate was higher in HRD + BRCAm tumors and in HRD + BRCAwt tumors (HRD + BRCAm: 97%, p = 0.004 and HRD + BRCAwt: 90%, p = 0.04) compared with 74% in the HRD-BRCAwt tumors. We also found Pt-sensitive patients tend to be enriched in patients with BRCA mutations or non-BRCA HRR pathway gene mutations (BRCA: 93.6% vs 75.4%, p < 0.001; non-BRCA HRR: 88.6% vs 75.4%, p = 0.062). Patients with HRD status positive had significantly improved PFS compared with those with HRD status negative (median PFS: 30.5 months vs. 16.8 months, Log-rank p = 0.001). Even for BRCAwt patients, positive HRD was also associated with better PFS than the HRD-negative group (median: 27.5 months vs 16.8 months, Log-rank p = 0.010). Further, we found patients with pathogenic mutations located in the DNA-binding domain (DBD) of BRCA1 had improved FPS, compared to those with mutations in other domains. (p = 0.03). CONCLUSIONS: The HRD status can be identified as an independent significance in Chinese HGSOC patients treated with first-line platinum-based chemotherapy.

Maize PPR-E proteins mediate RNA C-to-U editing in mitochondria by recruiting the trans deaminase PCW1.

RNA C-to-U editing in organelles is essential for plant growth and development; however, the underlying mechanism is not fully understood. Here, we report that pentatricopeptide repeat (PPR)-E subclass proteins carry out RNA C-to-U editing by recruiting the trans deaminase PPR motifs, coiled-coil, and DYW domain-containing protein 1 (PCW1) in maize (Zea mays) mitochondria. Loss-of-function of bZIP and coiled-coil domain-containing PPR 1 (bCCP1) or PCW1 arrests seed development in maize. bCCP1 encodes a bZIP and coiled-coil domain-containing PPR protein, and PCW1 encodes an atypical PPR-DYW protein. bCCP1 is required for editing at 66 sites in mitochondria and PCW1 is required for editing at 102 sites, including the 66 sites that require bCCP1. The PCW1-mediated editing sites are exclusively associated with PPR-E proteins. bCCP1 interacts with PCW1 and the PPR-E protein Empty pericarp7 (EMP7). Two multiple organellar RNA editing factor (MORF) proteins, ZmMORF1 and ZmMORF8, interact with PCW1, EMP7, and bCCP1. ZmMORF8 enhanced the EMP7-PCW1 interaction in a yeast three-hybrid assay. C-to-U editing at the ccmFN-1553 site in maize required EMP7, bCCP1, and PCW1. These results suggest that PPR-E proteins function in RNA editing by recruiting the trans deaminase PCW1 and bCCP1, and MORF1/8 assist this recruitment through protein-protein interactions.

Maize Shrek1 encodes a WD40 protein that regulates pre-rRNA processing in ribosome biogenesis.

Ribosome biogenesis is a fundamental and highly orchestrated process that involves hundreds of ribosome biogenesis factors. Despite advances that have been made in yeast, the molecular mechanism of ribosome biogenesis remains largely unknown in plants. We uncovered a WD40 protein, Shrunken and Embryo Defective Kernel 1 (SHREK1), and showed that it plays a crucial role in ribosome biogenesis and kernel development in maize (Zea mays). The shrek1 mutant shows an aborted embryo and underdeveloped endosperm and embryo-lethal in maize. SHREK1 localizes mainly to the nucleolus and accumulates to high levels in the seed. Depleting SHREK1 perturbs pre-rRNA processing and causes imbalanced profiles of mature rRNA and ribosome. The expression pattern of ribosomal-related genes is significantly altered in shrek1. Like its yeast (Saccharomyces cerevisiae) ortholog Periodic tryptophan protein 1 (PWP1), SHREK1 physically interacts with ribosomal protein ZmRPL7a, a transient component of the PWP1-subcomplex involved in pre-rRNA processing in yeast. Additionally, SHREK1 may assist in the A3 cleavage of the pre-rRNA in maize by interacting with the nucleolar protein ZmPOP4, a maize homolog of the yeast RNase mitochondrial RNA-processing complex subunit. Overall, our work demonstrates a vital role of SHREK1 in pre-60S ribosome maturation, and reveals that impaired ribosome function accounts for the embryo lethality in shrek1.

Regulator of Chromosome Condensation 1-Domain Protein DEK47 Functions on the Intron Splicing of Mitochondrial Nad2 and Seed Development in Maize.

In flowering plants, mitochondrial genes contain approximately 20-26 introns. Splicing of these introns is essential for mitochondrial gene expression and function. Recent studies have revealed that both nucleus- and mitochondrion-encoded factors are required for intron splicing, but the mechanism of splicing remains largely unknown. Elucidation of the mechanism necessitates a complete understanding of the splicing factors. Here, we report the identification of a regulator of chromosome condensation 1 (RCC1)-domain protein DEK47 that is required for mitochondrial intron splicing and seed development in maize. Loss of function in Dek47 severely arrests embryo and endosperm development, resulting in a defective kernel (dek) phenotype. DEK47 harbors seven RCC1 domains and is targeted to mitochondria. Null mutation of DEK47 causes a deficiency in the splicing of all four nad2 introns, abolishing the production of mature nad2 transcript and resulting in the disassembly and severely reduced activity of mitochondrial complex I. In response, the expression of the alternative oxidase AOX2 is sharply increased in dek47. These results indicate that Dek47 is required for the splicing of all the nad2 introns in mitochondria, and essential for complex I assembly, and kernel development in maize.

Long read sequencing of Toona sinensis (A. Juss) Roem: A chromosome-level reference genome for the family Meliaceae.

Chinese mahogany (Toona sinensis) is a woody plant that is widely cultivated in China and Malaysia. Toona sinensis is important economically, including as a nutritious food source, as material for traditional Chinese medicine and as a high-quality hardwood. However, the absence of a reference genome has hindered in-depth molecular and evolutionary studies of this plant. In this study, we report a high-quality T. sinensis genome assembly, with scaffolds anchored to 28 chromosomes and a total assembled length of 596 Mb (contig N50 = 1.5 Mb and scaffold N50 = 21.5 Mb). A total of 34,345 genes were predicted in the genome after homology-based and de novo annotation analyses. Evolutionary analysis showed that the genomes of T. sinensis and Populus trichocarpa diverged ~99.1-103.1 million years ago, and the T. sinensis genome underwent a recent genome-wide duplication event at ~7.8 million years and one more ancient whole genome duplication event at ~71.5 million years. These results provide a high-quality chromosome-level reference genome for T. sinensis and confirm its evolutionary position at the genomic level. Such information will offer genomic resources to study the molecular mechanism of terpenoid biosynthesis and the formation of flavour compounds, which will further facilitate its molecular breeding. As the first chromosome-level genome assembled in the family Meliaceae, it will provide unique insights into the evolution of members of the Meliaceae.

Two Pentatricopeptide Repeat Proteins Are Required for the Splicing of nad5 Introns in Maize.

Mitochondrial genes in flowering plants contain predominantly group II introns that require precise splicing before translation into functional proteins. Splicing of these introns is facilitated by various nucleus-encoded splicing factors. Due to lethality of mutants, functions of many splicing factors have not been revealed. Here, we report the function of two P-type PPR proteins PPR101 and PPR231, and their role in maize seed development. PPR101 and PPR231 are targeted to mitochondria. Null mutation of PPR101 and PPR231 arrests embryo and endosperm development, generating empty pericarp and small kernel phenotype, respectively, in maize. Loss-of-function in PPR101 abolishes the splicing of nad5 intron 2, and reduces the splicing of nad5 intron 1. Loss-of-function in PPR231 reduces the splicing of nad5 introns 1, 2, 3 and nad2 intron 3. The absence of Nad5 protein eliminates assembly of complex I, and activates the expression of alternative oxidase AOX2. These results indicate that both PPR101 and PPR231 are required for mitochondrial nad5 introns 1 and 2 splicing, while PPR231 is also required for nad5 intron 3 and nad2 intron 3. Both genes are essential to complex I assembly, mitochondrial function, and maize seed development. This work reveals that the splicing of a single intron involves multiple PPRs.

Em pty 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.

Construction of a high-density genetic map with whole genome sequencing in Nicotiana tabacum L.

Tobacco (Nicotiana tabacum L.) is an essential commercial crop and an ideal model plant for biological mechanism studies. As an allopolyploid species, tobacco harbors a massive and complex genome, which makes the application of molecular markers complicated and challenging. In our study, we performed whole-genome sequencing of an intraspecific recombinant inbred line (RIL) population, a F1 generation and their parents. With the Nicotiana tabacum (K326 cultivar) genome as reference, a total of 45,081 markers were characterized to construct the genetic map, which spanned a genetic distance of 3486.78 cM. Evaluation of a two-dimensional heat map proved the high quality of the genetic map. We utilized these markers to anchor scaffolds and analyzed the ancestral genome origin of linkage groups (LGs). Furthermore, such a high-density genetic map will be applied for quantitative trait locus (QTL) detection, gene localization, genome-wide association studies (GWAS), and marker-assisted breeding in tobacco.

Global transcriptome analysis of different stages of preimplantation embryo development in river buffalo.

BACKGROUND: Water buffalo (Bubalus bubalis) are divided into river buffalo and swamp buffalo subspecies and are essential livestock for agriculture and the local economy. Studies on buffalo reproduction have primarily focused on optimal fertility and embryonic mortality. There is currently limited knowledge on buffalo embryonic development, especially during the preimplantation period. Assembly of the river buffalo genome offers a reference for omics studies and facilitates transcriptomic analysis of preimplantation embryo development (PED). METHODS: We revealed transcriptomic profile of four stages (2-cell, 8-cell, Morula and Blastocyst) of PED via RNA-seq (Illumina HiSeq4000). Each stage comprised three biological replicates. The data were analyzed according to the basic RNA-seq analysis process. Ingenuity analysis of cell lineage control, especially transcription factor (TF) regulatory networks, was also performed. RESULTS: A total of 21,519 expressed genes and 67,298 transcripts were predicted from approximately 81.94 Gb of raw data. Analysis of transcriptome-wide expression, gene coexpression networks, and differentially expressed genes (DEGs) allowed for the characterization of gene-specific expression levels and relationships for each stage. The expression patterns of TFs, such as POU5F1, TEAD4, CDX4 and GATAs, were elucidated across diverse time series; most TF expression levels were increased during the blastocyst stage, during which time cell differentiation is initiated. All of these TFs were involved in the composition of the regulatory networks that precisely specify cell fate. These findings offer a deeper understanding of PED at the transcriptional level in the river buffalo.

The mitochondrial pentatricopeptide repeat protein EMP12 is involved in the splicing of three nad2 introns and seed development in maize.

Plant mitochondrial genes contain cis- and trans-group II introns that must be spliced before translation. The mechanism by which these introns are spliced is not well understood. Several families of proteins have been implicated in the intron splicing, of which the pentatricopeptide repeat (PPR) proteins are proposed to confer the substrate binding specificity. However, very few PPRs are characterized. Here, we report the function of a P-type PPR protein, EMP12, and its role in seed development. EMP12 is targeted to mitochondria. Loss-of-function mutation in Emp12 severely arrests embryo and endosperm development, causing embryo lethality. The trans-splicing of mitochondrial nad2 intron 2 and cis-splicing of nad2 intron 4 are abolished, whereas the cis-splicing of nad2 intron 1 is reduced in emp12 mutants. As a result, complex I assembly is disrupted, and its activity is strongly reduced in the mutants. The expression of the alternative oxidase and several components of other mitochondrial complexes is increased, possibly in response to the defective complex I. These results suggest that Emp12 is required for the trans-splicing of nad2 intron 2 and cis-splicing of nad2 introns 1 and 4, and is important to complex I biogenesis, and embryogenesis and endosperm development in maize.

EMPTY PERICARP16 is required for mitochondrial nad2 intron 4 cis-splicing, complex I assembly and seed development in maize.

In higher plants, chloroplast and mitochondrial transcripts contain a number of group II introns that need to be precisely spliced before translation into functional proteins. However, the mechanism of splicing and the factors involved in this process are not well understood. By analysing a seed mutant in maize, we report here the identification of Empty pericarp16 (Emp16) that is required for splicing of nad2 intron 4 in mitochondria. Disruption of Emp16 function causes developmental arrest in the embryo and endosperm, giving rise to an empty pericarp phenotype in maize. Differentiation of the basal endosperm transfer layer cells is severely affected. Molecular cloning indicates that Emp16 encodes a P-type pentatricopeptide repeat (PPR) protein with 11 PPR motifs and is localized in the mitochondrion. Transcript analysis revealed that mitochondrial nad2 intron 4 splicing is abolished in the emp16 mutants, leading to severely reduced assembly and activity of complex I. In response, the mutant dramatically increases the accumulation of mitochondrial complex III and the expression of alternative oxidase AOX2. These results imply that EMP16 is specifically required for mitochondrial nad2 intron 4 cis-splicing and is essential for complex I assembly and embryogenesis and development endosperm in maize.

Empty pericarp7 encodes a mitochondrial E-subgroup pentatricopeptide repeat protein that is required for ccmFN editing, mitochondrial function and seed development in maize.

RNA editing, converting cytidines (C) to uridines (U) at specific sites in the transcripts of mitochondria and plastids, plays a critical role in organelle gene expression in land plants. Recently pentatricopeptide repeat (PPR) proteins were identified as site-specific recognition factors for RNA editing. In this study, we characterized an empty pericarp7 mutant (emp7) in Zea mays (maize), which confers an embryo-lethal phenotype. In emp7 mutants, mitochondrial functions are seriously perturbed, resulting in a strikingly reduced respiration rate. Emp7 encodes an E-subgroup PPR protein that is localized exclusively in the mitochondrion. Null mutation of Emp7 abolishes the C → U editing of ccmF(N) transcript solely at position 1553. CcmF(N) is coding for a subunit of heme lyase complex in the cytochrome c maturation pathway. The resulting Phe → Ser substitution in CcmF(N) leads to the loss of CcmF(N) protein and a strikingly reduced c-type cytochrome. Consequently, the mitochondrial cytochrome-linked respiratory chain is impaired as a result of the disassembly of complex III in the emp7 mutant. These results indicate that the PPR-E subgroup protein EMP7 is required for C → U editing of ccmF(N) -1553 at a position essential for cytochrome c maturation and mitochondrial oxidative phosphorylation, and hence is essential to embryo and endosperm development in maize.

Small kernel 1 encodes a pentatricopeptide repeat protein required for mitochondrial nad7 transcript editing and seed development in maize (Zea mays) and rice (Oryza sativa).

RNA editing modifies cytidines (C) to uridines (U) at specific sites in the transcripts of mitochondria and plastids, altering the amino acid specified by the DNA sequence. Here we report the identification of a critical editing factor of mitochondrial nad7 transcript via molecular characterization of a small kernel 1 (smk1) mutant in Zea mays (maize). Mutations in Smk1 arrest both the embryo and endosperm development. Cloning of Smk1 indicates that it encodes an E-subclass pentatricopeptide repeat (PPR) protein that is targeted to mitochondria. Loss of SMK1 function abolishes the C â†’ U editing at the nad7-836 site, leading to the retention of a proline codon that is edited to encode leucine in the wild type. The smk1 mutant showed dramatically reduced complex-I assembly and NADH dehydrogenase activity, and abnormal biogenesis of the mitochondria. Analysis of the ortholog in Oryza sativa (rice) reveals that rice SMK1 has a conserved function in C â†’ U editing of the mitochondrial nad7-836 site. T-DNA knock-out mutants showed abnormal embryo and endosperm development, resulting in embryo or seedling lethality. The leucine at NAD7-279 is highly conserved from bacteria to flowering plants, and analysis of genome sequences from many plants revealed a molecular coevolution between the requirement for C â†’ U editing at this site and the existence of an SMK1 homolog. These results demonstrate that Smk1 encodes a PPR-E protein that is required for nad7-836 editing, and this editing is critical to NAD7 function in complex-I assembly in mitochondria, and hence to embryo and endosperm development in maize and rice.

Empty pericarp5 encodes a pentatricopeptide repeat protein that is required for mitochondrial RNA editing and seed development in maize.

In flowering plants, RNA editing is a posttranscriptional mechanism that converts specific cytidines to uridines in both mitochondrial and plastidial transcripts, altering the information encoded by these genes. Here, we report the molecular characterization of the empty pericarp5 (emp5) mutants in maize (Zea mays). Null mutation of Emp5 results in abortion of embryo and endosperm development at early stages. Emp5 encodes a mitochondrion-targeted DYW subgroup pentatricopeptide repeat (PPR) protein. Analysis of the mitochondrial transcripts revealed that loss of the EMP5 function abolishes the C-to-U editing of ribosomal protein L16 at the rpl16-458 site (100% edited in the wild type), decreases the editing at nine sites in NADH dehydrogenase9 (nad9), cytochrome c oxidase3 (cox3), and ribosomal protein S12 (rps12), and surprisingly increases the editing at five sites of ATP synthase F0 subunit a (atp6), apocytochrome b (cob), nad1, and rpl16. Mutant EMP5-4 lacking the E+ and DYW domains still retains the substrate specificity and editing function, only at reduced efficiency. This suggests that the E+ and DYW domains of EMP5 are not essential to the EMP5 editing function but are necessary for efficiency. Analysis of the ortholog in rice (Oryza sativa) indicates that rice EMP5 has a conserved function in C-to-U editing of the rice mitochondrial rpl16-458 site. EMP5 knockdown expression in transgenics resulted in slow growth and defective seeds. These results demonstrate that Emp5 encodes a PPR-DYW protein that is required for the editing of multiple transcripts in mitochondria, and the editing events, particularly the C-to-U editing at the rpl16-458 site, are critical to the mitochondrial functions and, hence, to seed development in maize.