Vacuolar iron stores gated by NRAMP3 and NRAMP4 are the primary source of iron in germinating seeds

During seed germination, iron (Fe) stored in vacuoles is exported by the redundant NRAMP3 and NRAMP4 transporter proteins. A double nramp3 nramp4 mutant is unable to mobilize Fe stores and does not develop in the absence of external Fe. We used RNA sequencing to compare gene expression in nramp3 nramp4 and wild type during germination and early seedling development. Even though sufficient Fe was supplied, the Fe-responsive transcription factors bHLH38, 39, 100, and 101 and their downstream targets FRO2 and IRT1 mediating Fe uptake were strongly upregulated in the nramp3 nramp4 mutant. Activation of the Fe deficiency response was confirmed by increased ferric chelate reductase activity in the mutant. At early stages, genes important for chloroplast redox control (FSD1 and SAPX), Fe homeostasis (FER1 and SUFB), and chlorophyll metabolism (HEMA1 and NYC1) were downregulated, indicating limited Fe availability in plastids. In contrast, expression of FRO3, encoding a ferric reductase involved in Fe import into the mitochondria, was maintained, and Fe-dependent enzymes in the mitochondria were unaffected in nramp3 nramp4. Together, these data show that a failure to mobilize Fe stores during germination triggered Fe deficiency responses and strongly affected plastids, but not mitochondria

Quantitative Analyses of the Yeast Oxidative Protein Folding Pathway in Vitro and in Vivo

Aims: Efficient oxidative protein folding (OPF) in the endoplasmic reticulum (ER) is a key requirement of the eukaryotic secretory pathway. In particular, protein folding linked to the formation of disulfide bonds, an activity dependent on the enzyme protein disulfide isomerase (PDI), is crucial. For the de novo formation of disulphide bonds, reduced PDI must be re-oxidised by an ER-located oxidase (ERO1). Despite some knowledge of this pathway, the kinetic parameters with which these components act and the importance of specific parameters, such as PDI reoxidation by Ero1, for the overall performance of OPF in vivo remain poorly understood. Results: We established an in vitro system using purified yeast (Saccharomyces cerevisiae) PDI (Pdi1p) and ERO1 (Ero1p) to investigate OPF. This necessitated the development of a novel reduction/oxidation processing strategy to generate homogenously oxidised recombinant yeast Ero1p. This new methodology enabled the quantitative assessment of the interaction of Pdi1p and Ero1p in vitro by measuring oxygen consumption and reoxidation of reduced RNAase A. The resulting quantitative data were then used to generate a simple model which can describe the oxidising capacity of Pdi1p and Ero1p in vitro and predict the in vivo effect of modulation of the levels of these proteins. Innovation: We describe a model that can be used to explore the OPF pathway and its control in a quantitative way. Conclusion: Our study provides new insights into how OPF works at a molecular level and provides a platform for the design of more efficient heterologous protein expression systems in yeast

The development of extension and magmatism in the Red Sea rift of Afaf

Despite the importance of continental breakup in plate tectonics, precisely how extensional processes such as brittle faulting, ductile plate stretching, and magma intrusion evolve in space and time during the development of new ocean basins remains poorly understood. The rifting of Arabia from Africa in the Afar depression is an ideal natural laboratory to address this problem since the region exposes subaerially the tectonically active transition from continental rifting to incipient seafloor spreading. We review recent constraints on along-axis variations in rift morphology, crustal and mantle structure, the distribution and style of ongoing faulting, subsurface magmatism and surface volcanism in the Red Sea rift of Afar to understand processes ultimately responsible for the formation of magmatic rifted continental margins. Our synthesis shows that there is a fundamental change in rift morphology from central Afar northward into the Danakil depression, spatially coincident with marked thinning of the crust, an increase in the volume of young basalt flows, and subsidence of the land towards and below sea-level. The variations can be attributed to a northward increase in proportion of extension by ductile plate stretching at the expense of magma intrusion. This is likely in response to a longer history of localised heating and weakening in a narrower rift. Thus, although magma intrusion accommodates strain for a protracted period during rift development, the final stages of breakup are dominated by a phase of plate stretching with a shift from intrusive to extrusive magmatism. This late-stage pulse of decompression melting due to plate thinning may be responsible for the formation of seaward dipping reflector sequences of basalts and sediments, which are ubiquitous at magmatic rifted margins worldwide

Loss of Cytochrome c Oxidase Promotes RAS-Dependent ROS Production from the ER Resident NADPH Oxidase, Yno1p, in Yeast.

Many disease states, including the aging process, are associated with the accumulation of mitochondria harboring respiratory dysfunction. Mitochondrial dysfunction is often accompanied by increased ROS levels that can contribute to cellular dysfunction and disease etiology. Here we use the model eukaryote S. cerevisiae to investigate whether reduced cytochrome c oxidase (COX) activity, commonly reported in aging organisms and associated with neurodegenerative disorders, leads to ROS production from mitochondria. We provide evidence that although reduced COX complex activity correlates with ROS accumulation, mitochondria are not the major production center. Instead we show that COX-deficient mitochondria activate Ras upon their outer membrane that establishes a pro-ROS accumulation environment by suppressing antioxidant defenses and the ERAD-mediated turnover of the ER-localized NADPH oxidase Yno1p. Our data suggest that dysfunctional mitochondria can serve as a signaling platform to promote the loss of redox homeostasis, ROS accumulation, and accelerate aging in yeast

Structural characterization and redox catalytic properties of cerium(IV) pyrochlore oxides

Ce(IV) pyrochlore oxides have been prepared by hydrothermal synthesis, and the parent material, a sodium cerium titanate, has been studied using total neutron scattering. While analysis of Bragg diffraction is consistent with an average cubic pyroclore structure, the profile is broadened because of the crystal size of <10 nm. Analysis of the pair distribution function (PDF) produced by Fourier transformation of the total scattering yields a structural model consistent with formulation of the parent material as (Na0.33Ce0.53Ti0.14)2Ti2O7. This contains a proportion of A-site titanium, consistent with the measured bulk density of the material. The PDF also contains evidence that the short-range order of the pyrochlore structure is disordered, with oxide anions displaced from the positions of the ideal Fd3̅m pyrochlore structure to give local symmetry F4̅3m. These observations are supported by static (broadline) solid state 49Ti NMR measurements on a 49Ti isotopically enriched sample, which showed a dominant, narrow resonance at an apparent shift of δ – 912 ppm and a second minor resonance consistent with A-site titanium. Sn(IV) doping of the pyrochlore phase is possible by one-step hydrothermal synthesis: this gives a series of materials with a maximum tin content of Sn:Ti = 0.4:0.6, for which 119Sn solid-state NMR confirms the presence of octahedral, B-site Sn(IV), and powder X-ray diffraction shows an associated expansion of the pyrochlore lattice. Temperature programmed reduction/oxidation studies of the materials reveal that after an activation cycle the parent pyrochlore shows a reversible low temperature reduction at <200 °C, more facile than ceria itself. The Sn-doped analogues also show a low temperature reduction, but on continued heating collapse irreversibly to yield a mixture of products that includes SnO. The parent pyrochlore has been tested as a support for gold in the water gas shift reaction and shows a lower temperature conversion of H2O and CO to H2 and CO2 than a ceria sample of similar surface area