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Ribosome assembly factors guide the complex process by which ribosomal proteins and the ribosomal RNAs form a functional ribosome. However, the assembly of plant plastid ribosomes is poorly understood. In the present study, we discovered a maize (Zea mays) plastid ribosome assembly factor based on our characterization of the embryo defective 15 (emb15) mutant. Loss of function of Emb15 retards embryo development at an early stage, but does not substantially affect the endosperm, and causes an albino phenotype in other genetic backgrounds. EMB15 localizes to plastids and possesses a ribosome maturation factor M (RimM) domain in the N-terminus and a predicted UDP-GlcNAc pyrophosphorylase domain in the C-terminus. The EMB15 RimM domain originated in bacteria and the UDP-GlcNAc pyrophosphorylase domain originated in fungi; these two domains came together in the ancestor of land plants during evolution. The N-terminus of EMB15 complemented the growth defect of an Escherichia coli strain with a RimM deletion and rescued the albino phenotype of emb15 homozygous mutants. The RimM domain mediates the interaction between EMB15 and the plastid ribosomal protein PRPS19. Plastid 16S rRNA maturation is also significantly impaired in emb15. These observations suggest that EMB15 functions in maize seed development as a plastid ribosome assembly factor, and the C-terminal domain is not important under normal conditions., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

The self-splicing of group II introns during RNA processing depends on their catalytic structure and is influenced by numerous factors that promote the formation of that structure through direct binding. Here we report that C-to-U editing at a specific position in two nad7 introns is essential to splicing, which also implies that the catalytic activity of non-functional group II introns could be restored by editing. We characterized a maize (Zea mays) mutant, dek46, with a defective kernel phenotype; Dek46 encodes a pentatricopeptide repeat DYW protein exclusively localized in mitochondria. Analyses of the coding regions of mitochondrial transcripts did not uncover differences in RNA editing between dek46 mutant and wild-type maize, but showed that splicing of nad7 introns 3 and 4 is severely reduced in the mutant. Furthermore, editing at nucleotide 22 of domain 5 (D5-C22) of both introns is abolished in dek46. We constructed chimeric introns by swapping D5 of P.li.LSUI2 with D5 of nad7 intron 3. In vitro splicing assays indicated that the chimeric intron containing D5-U22 can be self-spliced, but the one containing D5-C22 cannot. These results indicate that DEK46 functions in the C-to-U editing of D5-C22 of both introns, and the U base at this position is critical to intron splicing., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Splicing of plant organellar group II introns is under accurate nuclear control that employs many nucleus-encoded protein cofactors from various families. For mitochondrial introns, only a few splicing factors have been characterized because disruption of their functions often causes embryo lethality. Here, we report the function of Empty Pericarp8 (Emp8) in the splicing of three group II introns in mitochondria, complex I biogenesis, and seed development in maize. Emp8 encodes a P subfamily pentatricopeptide repeat protein that localizes in mitochondria. The loss-of-function mutants of Emp8 are embryo lethal, showing severely arrested embryo and endosperm development in maize. The respiration rate in the emp8 mutants is reduced with substantially enhanced expression of alternative oxidases. Transcript analysis indicated that the trans-splicing of nad1 intron 4 and cis-splicing of nad4 intron 1 are abolished, and the cis-splicing of nad2 intron 1 is severely impaired in the emp8 mutants. These defects consequently lead to the disassembly of mitochondrial complex I and a dramatic reduction in its activity. Together, these results suggest that Emp8 is required for the trans-splicing of nad1 intron 4 and cis-splicing of nad4 intron 1 and nad2 intron 1, which is essential to mitochondrial complex I assembly and hence to embryogenesis and endosperm development in maize., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

In higher plants, many mitochondrial genes contain group II-type introns that are removed from RNAs by splicing to produce mature transcripts that are then translated into functional proteins. However, the factors involved in the splicing of mitochondrial introns and their biological functions are not well understood in maize. Here, we isolated an empty pericarp 10 (emp10) mutant and identified the underlying gene by map-based cloning. Emp10 encodes a P-type mitochondria-targeted pentatricopeptide repeat (PPR) protein with 10 PPR motifs. Loss of Emp10 function results in splicing defect of the first intron of nad2, a gene encoding subunit 2 of NADH dehydrogenase (also called complex I). The emp10 mutant has undetectable activity of complex I and has arrested development of embryo and endosperm, and thus defective seeds with empty pericarp. Additionally, the basal endosperm transfer layer cells were severely affected, indicating the deficiency of cell wall ingrowths in the emp10 kernels. Moreover, the alternative respiratory pathway involving alternative oxidase was significantly induced in the emp10 mutant. These results suggest that EMP10 is specifically required for the cis-splicing of mitochondrial nad2 intron 1, embryogenesis and endosperm development in maize., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.)

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., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)

The embryo defective (emb) mutants in maize genetically define a unique class of loci that is required for embryogenesis but not endosperm development, allowing dissection of two developmental processes of seed formation. Through characterization of the emb14 mutant, we report here that Emb14 gene encodes a circular permuted, YqeH class GTPase protein that likely functions in 30S ribosome formation in plastids. Loss of Emb14 function in the null mutant arrests embryogenesis at the early transition stage. Emb14 was cloned by transposon tagging and was confirmed by analysis of four alleles. Subcellular localization indicated that the EMB14 is targeted to chloroplasts. Recombinant EMB14 is shown to hydrolyze GTP in vitro (Km  = 2.42 ± 0.3 μm). Emb14 was constitutively expressed in all tissues examined and high level of expression was found in transition stage embryos. Comparison of emb14 and WT indicated that loss of EMB14 function severely impairs accumulation of 16S rRNA and several plastid encoded ribosomal genes. We show that an EMB14 transgene complements the pale green, slow growth phenotype conditioned by mutations in AtNOA1, a closely related YqeH GTPase of Arabidopsis. Taken together, we propose that the EMB14/AtNOA1/YqeH class GTPases function in assembly of the 30S subunit of the chloroplast ribosome, and that this function is essential to embryogenesis in plants., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

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., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

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 → 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 → 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 → 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., (© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.)

Embryo-specific mutants in maize define a unique class of genetic loci that affect embryogenesis without a significant deleterious impact on endosperm development. Here we report the characterization of an embryo specific12 (emb12) mutant in maize. Embryogenesis in the emb12 mutants is arrested at or before transition stage. The mutant embryo at an early stage exhibits abnormal cell structure with increased vacuoles and dramatically reduced internal membrane organelles. In contrast, the mutant endosperm appears normal in morphology, cell structure, starch, lipid and protein accumulation. The Emb12 locus was cloned by transposon tagging and predicts a protein with a high similarity to prokaryotic translation initiation factor 3 (IF3). EMB12-GFP fusion analysis indicates that EMB12 is localized in plastids. The RNA in situ hybridization and protein immunohistochemical analyses indicate that a high level of Emb12 expression localizes in the embryo proper at early developmental stages and in the embryo axis at later stages. Western analysis indicates that plastid protein synthesis is impaired. These results indicate that Emb12 encodes the plastid IF3 which is essential for embryogenesis but not for endosperm development in maize., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

We implement a novel strategy for harnessing the power of high-copy transposons for functional analysis of the maize genome, and report behavioral features of the Mutator system in a uniform inbred background. The unique UniformMu population and database facilitate high-throughput molecular analysis of Mu-tagged mutants and gene knockouts. Key features of the population include: (i) high mutation frequencies (7% independent seed mutations) and moderation of copy number (approximately 57 total Mu elements; 1-2 MuDR copies per plant) were maintained by continuous back-crossing into a phenotypically uniform inbred background; (ii) a bz1-mum9 marker enabled selection of stable lines (loss of MuDR), inhibiting further transpositions in lines selected for molecular analysis; (iii) build-up of mutation load was prevented by screening Mu-active parents to exclude plants carrying pre-existing seed mutations. To create a database of genomic sequences flanking Mu insertions, selected mutant lines were analyzed by sequencing of MuTAIL PCR clone libraries. These sequences were annotated and clustered to facilitate bioinformatic subtraction of ancestral elements and identification of insertions unique to mutant lines. New insertions targeted low-copy, gene-rich sequences, and in silico mapping revealed a random distribution of insertions over the genome. Our results indicate that Mu populations differ markedly in the occurrence of Mu insertion hotspots and the frequency of suppressible mutations. We suggest that controlled MuDR copy number in UniformMu lines is a key determinant of these differences. The public database (http://uniformmu.org; http://endosperm.info) includes pedigree and phenotypic data for over 2000 independent seed mutants selected from a population of 31 548 F2 lines and integrated with analyses of 34 255 MuTAIL sequences.

A key regulated step in abscisic acid (ABA) biosynthesis in plants is catalyzed by 9-cis epoxycarotenoid dioxygenase (NCED), which cleaves 9-cis xanthophylls to xanthoxin, a precursor of ABA. In Arabidopsis, ABA biosynthesis is controlled by a small family of NCED genes. Nine carotenoid cleavage dioxygenase (CCD) genes have been identified in the complete genome sequence. Of these, five AtNCEDs (2, 3, 5, 6, and 9) have been cloned and studied for expression and subcellular localization. Although all five AtNCEDs are targeted to plastids, they differ in binding activity of the thylakoid membrane. AtNCED2, AtNCED3, and AtNCED6 are found in both stroma and thylakoid membrane-bound compartments. AtNCED5 is exclusively bound to thylakoids, whereas AtNCED9 remains soluble in stroma. A quantitative real-time PCR analysis and histochemical staining of promoter::GUS activity in transgenic Arabidopsis revealed a complex pattern of localized NCED expression in well-watered plants during development. AtNCED2 and AtNCED3 account for the NCED activity in roots, with localized expression in root tips, pericycle, and cortex cells at the base of lateral roots. Localized AtNCED2 and AtNCED3 expression in pericycle cells is an early marker of lateral initiation sites. AtNCED5, AtNCED6, AtNCED3, and AtNCED2 are expressed in flowers with very high AtNCED6::GUS activity occurring in pollen. AtNCED5::GUS, and to lesser degrees, AtNCED2::GUS and AtNCED3::GUS are expressed in developing anthers. AtNCED5, AtNCED6, AtNCED9, and AtNCED3 contribute to expression in developing seeds with high levels of AtNCED6 present at an early stage. GUS analysis indicates that AtNCED3 expression is confined to the base of the seed, whereas AtNCED5 and AtNCED6 are expressed throughout the seed. Consistent with the studies conducted by Iuchi and his colleagues in 2001, AtNCED3 is the major stress-induced NCED in leaves. Our results indicate that developmental control of ABA synthesis involves localized patterns of AtNCED gene expression. In addition, differential membrane-binding capacity of AtNCEDs is a potential means of post-translational regulation of NCED activity.