Embryo defective12 encodes the plastid initiation factor 3 and is essential for embryogenesis in maize

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.     

Emb15 encodes a plastid ribosomal assembly factor essential for embryogenesis in maize

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.     

The structure of YqeH. An AtNOS1/AtNOA1 ortholog that couples GTP hydrolysis to molecular recognition

AtNOS1/AtNOA1 was identified as a nitric oxide-generating enzyme in plants, but that function has recently been questioned. To resolve issues surrounding AtNOA1 activity, we report the biochemical properties and a 2.36 A resolution crystal structure of a bacterial AtNOA1 ortholog (YqeH). Geobacillus YqeH fused to a putative AtNOA1 leader peptide complements growth and morphological defects of Atnoa1 mutant plants. YqeH does not synthesize nitric oxide from L-arginine but rather hydrolyzes GTP. The YqeH structure reveals a circularly permuted GTPase domain and an unusual C-terminal beta-domain. A small N-terminal domain, disordered in the structure, binds zinc. Structural homology among the C-terminal domain, the RNA-binding regulator TRAP, and the hypoxia factor pVHL define a recognition module for peptides and nucleic acids. TRAP residues important for RNA binding are conserved by the YqeH C-terminal domain, whose positioning is coupled to GTP hydrolysis. YqeH and AtNOA1 probably act as G-proteins that regulate nucleic acid recognition and not as nitric-oxide synthases.     

AtNOS/AtNOA1 is a functional Arabidopsis thaliana cGTPase and not a nitric-oxide synthase

AtNOS1 was previously identified as a potential nitric-oxide synthase (NOS) in Arabidopsis thaliana, despite lack of sequence similarity to animal NOSs. Although the dwarf and yellowish leaf phenotype of Atnos1 knock-out mutant plants can be rescued by treatment with exogenous NO, doubts have recently been raised as to whether AtNOS1 is a true NOS. Moreover, depending on the type of physiological responses studied, Atnos1 is not always deficient in NO induction and/or detection, as previously reported. Here, we present experimental evidence showing that AtNOS1 is unable to bind and oxidize arginine to NO. These results support the argument that AtNOS1 is not a NOS. We also show that the renamed NO-associated protein 1 (AtNOA1) is a member of the circularly permuted GTPase family (cGTPase). AtNOA1 specifically binds GTP and hydrolyzes it. Complementation experiments of Atnoa1 mutant plants with different constructs of AtNOA1 show that GTP hydrolysis is necessary but not sufficient for the physiological function of AtNOA1. Mutant AtNOA1 lacking the C-terminal domain, although retaining GTPase activity, failed to complement Atnoa1, suggesting that this domain plays a crucial role in planta. cGTPases appear to be RNA-binding proteins, and the closest homolog of AtNOA1, the Bacillus subtilis YqeH, has been shown to participate in ribosome assembly and stability. We propose a similar function for AtNOA1 and discuss it in the light of its potential role in NO accumulation and plant development.     

Uncovering the Post-embryonic Role of Embryo Essential Genes in Arabidopsis using the Controlled Induction of Visibly Marked Genetic Mosaics: EMB506, an Illustration

The embryo essential gene EMB506 plays a crucial role in the transition of the Arabidopsis embryo from radial symmetry to bilateral symmetry just prior to the early heart stage of development. In addition to influencing embryo development EMB506 also affects chloroplast biogenesis. To further investigate the role of EMB506 gene expression in Arabidopsis we have generated green fluorescent protein (GFP) marked emb506 mosaic sectors at temporally defined stages during embryogenesis and additionally during various stages of vegetative growth, in otherwise phenotypically wild-type plants. We confirm the essential requirement for EMB506 gene expression in chloroplast biogenesis as reflected by the decreased chlorophyll content in emb506 mosaic sectors. We also show that the influence of EMB506 gene expression as it impinges on chloroplast biogenesis is first relevant at an intermediate stage in embryogenesis and that the role of EMB506 gene expression in chloroplast biogenesis is distinct from the essential role of EMB506 gene expression during early embryo development. By inducing emb506 mosaicism after the essential requirement for EMB506 gene expression in embryogenesis and also during vegetative growth we reveal that EMB506 gene expression additionally is required for correct cotyledon-, true leaf- and cauline leaf margin development. The strategy that we describe can be tailored to the mosaic analysis of any cloned EMB gene for which a corresponding mutant exists and can be applied to the mosaic analysis of mutant lethal genes in general.     

The GTP-binding protein YqeH participates in biogenesis of the 30S ribosome subunit in Bacillus subtilis

Bacterial genome sequencing has revealed a novel family of P-loop GTPases that are often essential for growth. Accumulating evidence suggests that these proteins are involved in biogenesis of the 30S or 50S ribosomal subunits. YqeH is a member of this Obg/Era GTPase family, with its function remains to be uncovered. Here, we present results showing that YqeH is involved in the 30S subunit biogenesis in Bacillus subtilis. We observed a reduction in the 70S ribosome and accumulation of the free 50S subunit in YqeH-depleted cells. Interestingly, no free 30S subunit accumulation was evident. Consistent with the theory that YqeH is involved in 30S subunit biogenesis, a precursor of 16S rRNA and its degradation products were detected. Additionally, the reduction of free 30S subunit was not observed in Era-depleted cells. YqeH overexpression did not compensate for growth defects in mutants devoid of Era and vice versa. Moreover, in vitro GTPase analyses showed that YqeH possessed high intrinsic GTPase activity. In contrast, Era showed slow GTPase activity, which was enhanced by the 30S ribosomal subunit. Our findings strongly suggest that YqeH and Era function at distinct checkpoints during 30S subunit assembly. B. subtilis yqeH is classified as an essential gene due to the inability of the IPTG-dependent P(spac)-yqeH mutant to grow on LB or PAB agar plates in the absence of IPTG. However, in our experiments, the P(spac)-yqeH mutant grew in PAB liquid medium without IPTG supplementation, albeit at an impaired rate. This finding raises the interesting possibility that YqeH participates in assembly of the 30S ribosomal subunit as well as other cellular functions essential for growth on solid media.     

EMB1211 is required for normal embryo development and influences chloroplast biogenesis in Arabidopsis

Chloroplast biogenesis is tightly linked with embryogenesis and seedling development. A growing body of work has been done on the molecular mechanisms underlying chloroplast development; however, the molecular components involved in chloroplast biogenesis during embryogenesis remain largely uncharacterized. In this paper, we show that an Arabidopsis mutant carrying a T‐DNA insertion in a gene encoding a multiple membrane occupation and recognition nexus (MORN)‐containing protein exhibits severe defects during embryogenesis, producing abnormal embryos and thereby leading to a lethality of young seedlings. Genetic and microscopic studies reveal that the mutation is allelic to a previously designated Arabidopsis embryo‐defective 1211 mutant (emb1211). The emb1211 +/? mutant plants produce approximately 25% of white‐colored ovules with abnormal embryos since late globular stage when primary chloroplast biogenesis takes place, while the wild‐type plants produce all green ovules. Transmission electron microscopic analysis reveals the absence of normal chloroplast development, both in the mutant embryos and in the mutant seedlings, that contributes to the albinism. The EMB1211 gene is preferentially expressed in developing embryos as revealed in the EMB1211::GUS transgenic plants. Taken together, the data indicate that EMB1211 has an important role during embryogenesis and chloroplast biogenesis in Arabidopsis.     

The Requirement of WHIRLY1 for Embryogenesis Is Dependent on Genetic Background in Maize

Plastid gene expression is essential to embryogenesis in higher plants, but the underlying mechanism is obscure. Through molecular characterization of an embryo defective 16 (emb16) locus, here we report that the requirement of plastid translation for embryogenesis is dependent on the genetic background in maize (Zea mays). The emb16 mutation arrests embryogenesis at transition stage and allows the endosperm to develop largely normally. Molecular cloning reveals that Emb16 encodes WHIRLY1 (WHY1), a DNA/RNA binding protein that is required for genome stability and ribosome formation in plastids. Interestingly, the previous why1 mutant alleles (why1-1 and why1-2) do not affect embryogenesis, only conditions albino seedlings. The emb16 allele of why1 mutation is in the W22 genetic background. Crosses between emb16 and why1-1 heterozygotes resulted in both defective embryos and albino seedlings in the F1 progeny. Introgression of the emb16 allele from W22 into A188, B73, Mo17, Oh51a and the why1-1 genetic backgrounds yielded both defective embryos and albino seedlings. Similar results were obtained with two other emb mutants (emb12 and emb14) that are impaired in plastid protein translation process. These results indicate that the requirement of plastid translation for embryogenesis is dependent on genetic backgrounds, implying a mechanism of embryo lethality suppression in maize.     

Circularly permuted GTPase YqeH binds 30S ribosomal subunit: Implications for its role in ribosome assembly

YqeH, a circularly permuted GTPase, is conserved among bacteria and eukaryotes including humans. It was shown to be essential for the assembly of small ribosomal (30S) subunit in bacteria. However, whether YqeH interacts with 30S ribosome and how it may participate in 30S assembly are not known. Here, using co-sedimentation experiments, we report that YqeH co-associates with 30S ribosome in the GTP-bound form. In order to probe whether YqeH functions as RNA chaperone in 30S assembly, we assayed for strand dissociation and annealing activity. While YqeH does not exhibit these activities, it binds a non-specific single and double-stranded RNA, which unlike the 30S binding is independent of GTP/GDP binding and does not affect intrinsic GTP hydrolysis rates. Further, S5, a ribosomal protein which participates during the initial stages of 30S assembly, was found to promote GTP hydrolysis and RNA binding activities of YqeH.     

The essential GTPase YqeH is required for proper ribosome assembly in Bacillus subtilis

Recent work with bacteria and eukaryotes has shown that GTPases play important roles in ribosome assembly. Here we show that the essential GTPase YqeH is required for proper 70S ribosome formation and 30S subunit assembly/stability in Bacillus subtilis.     

In vivo functional characterization of the <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> 60S biogenesis GTPase Nog1

The Saccharomyces cerevisiae Nog1 GTPase is critical for assembly of the large ribosomal subunit. Mutations in conserved residues in the GTP-binding pocket cause defects in cell growth and 60S ribosome assembly but mutant proteins retain their ability to associate with the pre-60S. Association of Nog1 with the pre-60S is independent of guanine nucleotide added to cell extracts. Thus, it appears that nucleotide occupancy does not substantially affect Nog1 association with pre-60S particles. Somewhat surprisingly, neither of the conserved threonines in the G2 motif of the GTPase domain is essential for Nog1 function. Neither the steady-state rRNA levels nor the protein composition (as determined by isobaric labeling and identification by mass spectrometry of peptides) of the pre-60S particles in the nog1P176V mutant are grossly perturbed, although levels of four proteins (Nog1, Nop2, Nop15, and Tif6) are modestly reduced in pre-60S particles isolated from the mutant. Deletion analysis revealed that the C-terminal 168 amino acids are not required for function; however, the N-terminal 126 amino acids are required. Optimal association with pre-60S particles requires sequences between amino acids 347–456. Several conserved charge-to-alanine substitutions outside the GTPase domain display modest growth phenotypes indicating that these residues are not critical for function. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Deciphering the Catalytic Machinery in 30S Ribosome Assembly GTPase YqeH

Background

YqeH, a circularly permuted GTPase (cpGTPase), which is conserved across bacteria and eukaryotes including humans is important for the maturation of small (30S) ribosomal subunit in Bacillus subtilis. Recently, we have shown that it binds 30S in a GTP/GDP dependent fashion. However, the catalytic machinery employed to hydrolyze GTP is not recognized for any of the cpGTPases, including YqeH. This is because they possess a hydrophobic substitution in place of a catalytic glutamine (present in Ras-like GTPases). Such GTPases were categorized as HAS-GTPases and were proposed to follow a catalytic mechanism, different from the Ras-like proteins.

Methodology/Principal Findings

MnmE, another HAS-GTPase, but not circularly permuted, utilizes a potassium ion and water mediated interactions to drive GTP hydrolysis. Though the G-domain of MnmE and YqeH share only ∼25% sequence identity, the conservation of characteristic sequence motifs between them prompted us to probe GTP hydrolysis machinery in YqeH, by employing homology modeling in conjunction with biochemical experiments. Here, we show that YqeH too, uses a potassium ion to drive GTP hydrolysis and stabilize the transition state. However, unlike MnmE, it does not dimerize in the transition state, suggesting alternative ways to stabilize switches I and II. Furthermore, we identify a potential catalytic residue in Asp-57, whose recognition, in the absence of structural information, was non-trivial due to the circular permutation in YqeH. Interestingly, when compared with MnmE, helix α2 that presents Asp-57 is relocated towards the N-terminus in YqeH. An analysis of the YqeH homology model, suggests that despite such relocation, Asp-57 may facilitate water mediated catalysis, similarly as the catalytic Glu-282 of MnmE. Indeed, an abolished catalysis by D57I mutant supports this inference.

Conclusions/Significance

An uncommon means to achieve GTP hydrolysis utilizing a K⁺ ion has so far been demonstrated only for MnmE. Here, we show that YqeH also utilizes a similar mechanism. While the catalytic machinery is similar in both, mechanistic differences may arise based on the way they are deployed. It appears that K⁺ driven mechanism emerges as an alternative theme to stabilize the transition state and hydrolyze GTP in a subset of GTPases, such as the HAS-GTPases.     

Human C4orf14 interacts with the mitochondrial nucleoid and is involved in the biogenesis of the small mitochondrial ribosomal subunit

The bacterial homologue of C4orf14, YqeH, has been linked to assembly of the small ribosomal subunit. Here, recombinant C4orf14 isolated from human cells, co-purified with the small, 28S subunit of the mitochondrial ribosome and the endogenous protein co-fractionated with the 28S subunit in sucrose gradients. Gene silencing of C4orf14 specifically affected components of the small subunit, leading to decreased protein synthesis in the organelle. The GTPase of C4orf14 was critical to its interaction with the 28S subunit, as was GTP. Therefore, we propose that C4orf14, with bound GTP, binds to components of the 28S subunit facilitating its assembly, and GTP hydrolysis acts as the release mechanism. C4orf14 was also found to be associated with human mitochondrial nucleoids, and C4orf14 gene silencing caused mitochondrial DNA depletion. In vitro C4orf14 is capable of binding to DNA. The association of C4orf14 with mitochondrial translation factors and the mitochondrial nucleoid suggests that the 28S subunit is assembled at the mitochondrial nucleoid, enabling the direct transfer of messenger RNA from the nucleoid to the ribosome in the organelle.     

Partial complementation of embryo defective mutations: a general strategy to elucidate gene function

The EMB 506 gene has been characterised as essential for embryo development. To provide insights into the role of EMB 506, which is hidden by the embryo defective phenotype, the ABI3 promoter was fused to the EMB 506 cDNA. The expression of such a transgene should provide sufficient protein during embryogenesis to ensure normal embryo development in homozygous emb 506 seeds. We show that homozygous emb 506 seedlings, partially complemented with the ABI3::EMB 506 transgene, can be obtained. Most of the rescued emb 506 plants are able to flower and to set normal seeds, but show mild to severe depigmentation of rosette leaves and/or inflorescences. This effect on chloroplast development indicated a putative chloroplast localisation of the EMB 506 protein, which was demonstrated by GFP-protein fusion. However, EMB 506 cannot be considered as a chloroplast housekeeping protein only, since EMB 506 is not present in all photosynthetic tissues. This study demonstrates the power of this simple strategy, which could be widely applied to other emb mutants and which may reveal similar or additional roles for EMB genes at vegetative stages of the life cycle.     

182 Investigating the mode of action of an essential OBG GTPase,CgtA in bacteria

CgtA is an essential OBG GTPase (Trach & Hoch, 1989) highly conserved from bacteria to eukaryotes. It is a multifunctional protein, involved in DNA replication, chromosome partitioning (Slominska et al., 2002), nutritional stress response, initiation of sporulation, ribosome maturation, etc. Despite being a multifunctional essential protein, its mode of action is not well- characterized and key question remains: how does this protein work in wide varieties of cellular function? The expression of cgtA-mRNA increases on the onset of nutritional stress. Purified CgtA protein shows increased GTPase activity in the presence of ribosome. Our experiment with thiostrepton reveals that, although ribosome is able to trigger the GTPase activity of CgtA, its probable site of GTPase inducing activity is different from other regular translation factors like EF-G, that uses GTP. For structure function study we have generated an energy minimized homology model of the Vibrio cholerae CgtA protein, which reveals two large domains, an OBG-fold and a GTP– hydrolysis domain, with an extended C-terminal part. We compared the amino acid sequence of CgtA across various species in the database, and found that its Glycine98 and the Tyrosine195 residues are 100% conserved in prokaryotes. These amino acids are highly conserved in eukaryotes as well. Gly98 and Tyr195 are located on the hinge region of CgtA comprising of portions of the OBG and the GTP–hydrolysis domains, respectively. To decipher the mode of actions of CgtA and the role of the conserved Gly98 residue, we have replaced the Gly with a relatively larger amino acid, i.e. Asp. Our study reveals that the mutant CgtA(G98D) shows a reduced GTPase activity in presence of ribosome compared to the wild type. This indicates a restricted inter-domain movement of CgtA due to the above point mutation. To understand this phenomenon we are using MD simulations. We will discuss results from MD simulations and other mutation studies as well. Our results indicate that ribosome acts as a modulator for increasing the GTPase activity of CgtA. The perfect conservation of G98 residue is important for the proper functionality of CgtA.     

ULTRASTRUCTURE OF ARRESTED EMBRYOS FROM LETHAL MUTANTS OF ARABIDOPSIS THALIANA

The purpose of this study was to examine the extent of cellular differentiation in arrested embryos from lethal mutants of Arabidopsis thaliana. The question to be addressed was whether arrested embryos in heterozygous siliques resembled mature wild-type embryos at the cellular level. Protein bodies were chosen as developmental markers because they appear only during the final stages of embryogenesis. Both the hypocotyl and cotyledons of wild-type embryos contained protein bodies that became filled with storage protein during the cotyledonary stages of development. Some mutant embryos (emb30) contained normal protein bodies and resembled mature wild-type embryos at the cellular level. Other mutant embryos (emb22) contained only immature protein bodies and were therefore blocked in both morphogenesis and cellular differentiation. The formation of protein bodies in emb31 was normal in the hypocotyl but delayed in the cotyledons. In this case the mutant gene appears to disrupt the timing of both morphogenesis and differentiation. This ultrastructural view of arrested embryos has provided additional information on the nature of developmental arrest that should facilitate the classification of embryonic lethals and the identification of mutants with defects in developmental rather than housekeeping functions.     

A Bacterial GAP-Like Protein, YihI, Regulating the GTPase of Der, an Essential GTP-Binding Protein in Escherichia coli

Der (double Era-like GTPase) is an essential GTPase consisting of two GTP-binding motifs in tandem followed by a KH-like domain. Der plays a critical role in 50S ribosome maturation at a later biogenesis step. Here, we attempted to identify a protein interacting with Der that modulates its function and regulation. Using a yeast two-hybrid, we discovered that Der interacts with YihI, which activates the GTPase activity of Der. Its overexpression affected cell growth, causing accumulation of rRNA precursors and an aberrant ribosome profile that was similar to that of Der-depleted cells, suggesting that Der and YihI are involved in the 50S ribosome assembly. The yihI deletion strain showed a shorter lag phase than wild-type strain, suggesting that YihI may be a negative regulator for ribosome assembly. We propose that YihI is a GAP (GTPase-activating protein)-like protein that modulates Der function to negatively regulate cell growth at the beginning of exponential growth.