EMBRYONIC FACTOR 31 encodes a tyrosyl-tRNA synthetase that is essential for seed development

Aminoacyl-tRNA synthetases (AARSs) involve the process of catalyzing the ligation of specific amino acids to their cognate tRNAs. Here we identified an Arabidopsis mutant embryonic factor 31 (fac31), its embryos arrested at development from one cell to globular stage. The FAC31 gene was identified by positional cloning and confirmed by a genetic complementation test with two independent T-DNA insertion lines and transgenic rescue with full-length genomic DNA. FAC31 encodes a Tyrosyl-tRNA synthetase and localize to mitochondria and cytoplasm. Fac31 mutants contain a point mutation from CAA to a stop codon TAA which may lead to a truncated protein. The phenotype of fac31 mutants are very similar to the T-DNA insertion lines Salk_016722 and Salk_045570 displayed smaller embryo sac contains only less number of endosperm nucleolus. Genetic analysis showed that the FAC31 gene had no parental effects through the transmission of mutated FAC31 gene by gametes. FAC31 is a high-conserved protein among animals and plants. RT-PCR analysis and promoter-GUS expression showed that it is expressed in nearly all tissues tested, strongly expressed in meristem of seedlings, the primordium of lateral root, young inflorescences, mature pollen, germinated pollen tubes and embryo sacs before heart stage. Our findings suggest that FAC31 is essential for the seed development through regulation the expanding of embryo sac and proliferation of endosperm nucleolus.     

Biochemical and physiological investigations on adenosine 5ʹ monophosphate deaminase from Plasmodium spp.

The interplay between ATP generating and utilizing pathways in a cell is responsible for maintaining cellular ATP/energy homeostasis that is reflected by Adenylate Energy Charge (AEC) ratio. Adenylate kinase (AK), that catalyzes inter‐conversion of ADP, ATP and AMP, plays a major role in maintaining AEC and is regulated by cellular AMP levels. Hence, the enzymes AMP deaminase (AMPD) and nucleotidases, which catabolize AMP, indirectly regulate AK activity and in‐turn affect AEC. Here, we present the first report on AMPD from Plasmodium, the causative agent of malaria. The recombinant enzyme expressed in Saccharomyces cerevisiae was studied using functional complementation assay and residues vital for enzyme activity have been identified. Similarities and differences between Plasmodium falciparum AMPD (PfAMPD) and its homologs from yeast, Arabidopsis and humans are also discussed. The AMPD gene was deleted in the murine malaria parasite P. berghei and was found to be dispensable during all stages of the parasite life cycle. However, when episomal expression was attempted, viable parasites were not obtained, suggesting that perturbing AMP homeostasis by over‐expressing AMPD might be lethal. As AMPD is known to be allosterically modulated by ATP, GTP and phosphate, allosteric activators of PfAMPD could be developed as anti‐parasitic agents.     

GAMETOPHYTIC FACTOR 1, Involved in Pre-mRNA Splicing, Is Essential for Megagametogenesis and Embryogenesis in Arabidopsis

RNA biogenesis is essential and vital for accurate expression of genes. It is obvious that cells cannot continue normal metabolism when RNA splicing is interfered with. sgt13018 is such a mutant, with partial loss of function of GAMETOPHYTIC FACTOR 1 (GFA1); a gene likely involved in RNA biogenesis in Arabidopsis. The mutant is featured in the phenotype of diminished female gametophyte development at stage FG5 and is associated with the arrest of early embryo development in Arabidopsis. Bioinformatics data showed that homoiogs of gene GFA1 in yeast and human encode putative U5 snRNPspecific proteins required for pre-mRNA splicing. Furthermore, the result of yeast two-hybrid assay indicated that GFA1 physically interacted with AtBrr2 and AtPrp8, the putative U5 snRNP components, of Arabidopsis. This investigation suggests that GFA1 is involved in mRNA biogenesis through interaction with AtBrr2 and AtPrp8 and functions in megagametogeneeis and embryogenesis in plant.     

EMBRYONIC FACTOR 19 encodes a pentatricopeptide repeat protein that is essential for the initiation of zygotic embryogenesis in Arabidopsis

Early embryogenesis is the most fundamental developmental process in biology.Screening of ethyl methanesulfonate(EMS)-mutagenized populations of Arabidopsis thaliana led to the identification of a zygote-lethal mutant embryonic factor 19(fac19)in which embryo development was arrested at the elongated zygote to octant stage.The number of endosperm nuclei decreased significantly in fac19 embryos.Genetic analysis showed fac19 was caused by a single recessive mutation with typical mendelian segregation,suggesting equal maternal and paternal contributions of FAC19 towards zygotic embryogenesis.Positional cloning showed that FAC19 encodes a putative mitochondrial protein with 16 conserved pentatricopeptide repeat(PPR)motifs.The fac19 mutation caused a conversion from hydrophilic serine located in a previously unknown domain to hydrophobic leucine.Crosses between FAC19/fac19 and the T-DNA insertion mutants in the same gene failed to complement the fac19 defects,confirming the identity of the gene.This study revealed the critical importance of a PPR protein-mediated mitochondrial function in early embryogenesis.     

Genetic and other determinants of AMP deaminase activity in healthy adult skeletal muscle

AMPD1 genotype,relative fiber type composition, training status, and gender wereevaluated as contributing factors to the reported variation in AMPdeaminase enzyme activity in healthy skeletal muscle. Multifactorialcorrelative analyses demonstrate thatAMPD1 genotype has the greatest effecton enzyme activity. An AMPD1 mutantallele frequency of 13.7 and a 1.7% incidence of enzyme deficiency wasfound across 175 healthy subjects. Homozygotes for theAMPD1 normal allele have high enzymeactivities, and heterozygotes display intermediate activities. Whenexamined according to genotype, other factors were found to affectvariability as follows: AMP deaminase activity in homozygotes for thenormal allele exhibits a negative correlation with the relativepercentage of type I fibers and training status. Conversely, residualAMP deaminase activity in homozygotes for the mutant allele displays apositive correlation with the relative percentage of type I fibers.Opposing correlations in different homozygousAMPD1 genotypes are likely due torelative fiber-type differences in the expression ofAMPD1 andAMPD3 isoforms. Gender alsocontributes to variation in total skeletal muscle AMP deaminaseactivity, with normal homozygous and heterozygous women showing only85-88% of the levels observed in genotype-matched men.

     

Membrane association, mechanism of action, and structure of Arabidopsis embryonic factor 1 (FAC1)

Embryonic factor 1 (FAC1) is one of the earliest expressed plant genes and encodes an AMP deaminase (AMPD), which is also an identified herbicide target. This report identifies an N-terminal transmembrane domain in Arabidopsis FAC1, explores subcellular fractionation, and presents a 3.3-A globular catalytic domain x-ray crystal structure with a bound herbicide-based transition state inhibitor that provides the first glimpse of a complete AMPD active site. FAC1 contains an (alpha/beta)(8)-barrel characterized by loops in place of strands 5 and 6 that places it in a small subset of the amidohydrolase superfamily with imperfect folds. Unlike tetrameric animal orthologs, FAC1 is a dimer and each subunit contains an exposed Walker A motif that may be involved in the dramatic combined K(m) (25-80-fold lower) and V(max) (5-6-fold higher) activation by ATP. Normal mode analysis predicts a hinge motion that flattens basic surfaces on each monomer that flank the dimer interface, which suggests a reversible association between the FAC1 globular catalytic domain and intracellular membranes, with N-terminal transmembrane and disordered linker regions serving as the anchor and attachment to the globular catalytic domain, respectively.     

AMPD3 is involved in anthrax LeTx-induced macrophage cell death

The responses of macrophages to Bacillus anthracis infection are important for the survival of the host, since macrophages are required for the germination of B. anthracis spores in lymph nodes, and macrophage death exacerbates anthrax lethal toxin (LeTx)-induced organ collapse. To elucidate the mechanism of macrophage cell death induced by LeTx, we performed a genetic screen to search for genes associated with LeTx-induced macrophage cell death. RAW 264.7 cells, a macrophage-like cell line sensitive to LeTx-induced death, were randomly mutated and LeTx-resistant mutant clones were selected. AMP deaminase 3 (AMPD3), an enzyme that converts AMP to IMP, was identified to be mutated in one of the resistant clones. The requirement of AMPD3 in LeTx-induced cell death of RAW 264.7 cells was confirmed by the restoration of LeTx sensitivity with ectopic reconstitution of AMPD3 expression. AMPD3 deficiency does not affect LeTx entering cells and the cleavage of mitogen-activated protein kinase kinase (MKK) by lethal factor inside cells, but does impair an unknown downstream event that is linked to cell death. Our data provides new information regarding LeTx-induced macrophage death and suggests that there is a key regulatory site downstream of or parallel to MKK cleavage that controls the cell death in LeTx-treated macrophages.     

Metformin activates AMP kinase through inhibition of AMP deaminase

The mechanism for how metformin activates AMPK (AMP-activated kinase) was investigated in isolated skeletal muscle L6 cells. A widely held notion is that inhibition of the mitochondrial respiratory chain is central to the mechanism. We also considered other proposals for metformin action. As metabolic pathway markers, we focused on glucose transport and fatty acid oxidation. We also confirmed metformin actions on other metabolic processes in L6 cells. Metformin stimulated both glucose transport and fatty acid oxidation. The mitochondrial Complex I inhibitor rotenone also stimulated glucose transport but it inhibited fatty acid oxidation, independently of metformin. The peroxynitrite generator 3-morpholinosydnonimine stimulated glucose transport, but inhibited fatty acid oxidation. Addition of the nitric oxide precursor arginine to cells did not affect glucose transport. These studies differentiate metformin from inhibition of mitochondrial respiration and from active nitrogen species. Knockdown of adenylate kinase also failed to affect metformin stimulation of glucose transport. Hence, any means of increase in ADP appears not to be involved in the metformin mechanism. Knockdown of LKB1, an upstream kinase and AMPK activator, did not affect metformin action. Having ruled out existing proposals, we suggest a new one: metformin might increase AMP through inhibition of AMP deaminase (AMPD). We found that metformin inhibited purified AMP deaminase activity. Furthermore, a known inhibitor of AMPD stimulated glucose uptake and fatty acid oxidation. Both metformin and the AMPD inhibitor suppressed ammonia accumulation by the cells. Knockdown of AMPD obviated metformin stimulation of glucose transport. We conclude that AMPD inhibition is the mechanism of metformin action.     

Vitamer levels, stress response, enzyme activity, and gene regulation of Arabidopsis lines mutant in the pyridoxine/pyridoxamine 5'-phosphate oxidase (PDX3) and the pyridoxal kinase (SOS4) genes involved in the vitamin B6 salvage pathway

PDX3 and SALT OVERLY SENSITIVE4 (SOS4), encoding pyridoxine/pyridoxamine 5'-phosphate oxidase and pyridoxal kinase, respectively, are the only known genes involved in the salvage pathway of pyridoxal 5'-phosphate in plants. In this study, we determined the phenotype, stress responses, vitamer levels, and regulation of the vitamin B(6) pathway genes in Arabidopsis (Arabidopsis thaliana) plants mutant in PDX3 and SOS4. sos4 mutant plants showed a distinct phenotype characterized by chlorosis and reduced plant size, as well as hypersensitivity to sucrose in addition to the previously noted NaCl sensitivity. This mutant had higher levels of pyridoxine, pyridoxamine, and pyridoxal 5'-phosphate than the wild type, reflected in an increase in total vitamin B(6) observed through HPLC analysis and yeast bioassay. The sos4 mutant showed increased activity of PDX3 as well as of the B(6) de novo pathway enzyme PDX1, correlating with increased total B(6) levels. Two independent lines with T-DNA insertions in the promoter region of PDX3 (pdx3-1 and pdx3-2) had decreased PDX3 activity. Both also had decreased activity of PDX1, which correlated with lower levels of total vitamin B(6) observed using the yeast bioassay; however, no differences were noted in levels of individual vitamers by HPLC analysis. Both pdx3 mutants showed growth reduction in vitro and in vivo as well as an inability to increase growth under high light conditions. Increased expression of salvage and some of the de novo pathway genes was observed in both the pdx3 and sos4 mutants. In all mutants, increased expression was more dramatic for the salvage pathway genes.     

Functional complementation of yeast vma1 delta cells by a plant subunit A homolog rescues the mutant phenotype and partially restores vacuolar H(+)-ATPase activity

The ability of a vacuolar H(+)-ATPase (V-ATPase) subunit homolog (subunit A) from plants to rescue the vma mutant phenotype of yeast was investigated as a first step towards investigating the structure and function of plant subunits in molecular detail. Heterologous expression of cotton cDNAs encoding near-identical isoforms of subunit A in mutant vma1 delta yeast cells successfully rescued the mutant vma phenotype, indicating that subunit A of plants and yeast have retained elements essential to V-ATPases during the course of evolution. Although vacuoles become acidified, the plant-yeast hybrid holoenzyme only partially restored V-ATPase activity (approximately 60%) in mutant yeast cells. Domain substitution of divergent N- or C-termini only slightly enhanced V-ATPase activity, whereas swapping both domains acted synergistically, increasing coupled ATP hydrolysis and proton translocation by approximately 22% relative to the native plant subunit. Immunoblot analysis indicated that similar amounts of yeast, plant or plant-yeast chimeric subunits are membrane-bound. These results suggest that subunit A terminal domains contain structural information that impact V-ATPase structure and function.