Flux Analysis of Glucose Metabolism in Rhizopus oryzae for the Purpose of Increasing Lactate Yields

A flux analysis of glucose metabolism in the filamentous fungus Rhizopus oryzae was achieved using a specific radioactivity curve-matching program, TFLUX. Glycolytic and tricarboxylic acid cycle intermediates labeled through the addition of extracellular [U-14C]glucose were isolated and purified for specific radioactivity determinations. This information, together with pool sizes and the rates of glucose utilization and end product production, provided input for flux maps of the metabolic network under two different experimental conditions. Based upon the flux analysis of this system, a mutant of R. oryzae with higher lactate and lower ethanol yields than the parent was sought for and found.     

The Effect of Coumarin on Growth, Production of Dry Matter, Protein and Nucleic Acids in Roots of Maize and Wheat and the Interaction of Coumarin with Metabolic Inhibitors

Effects of coumarin on fresh weight, dry matter, protein and nucleic acid content per cell in attached roots of maize and wheat and in whole excised elongation zones of maize were determined. The inhibition in cell length exerted by coumarin did not correspond to an inhibition of the net synthetic capacity. Coumarin treatment increased the cell surface, the production of dry matter and the protein content per cell. The dry matter and the protein content per unit surface was slightly increased or unaffected. The effect of coumann on cell shape seemed to be independent of that on dry matter production and net protein synthesis. The same was found in excised elongation zones. —The net DNA-synthesis per cell was slightly increased in attached roots by coumann treatment, but this effect was probably not correlated with the morphogenetic changes. Inhibition of DNA-synthesis with hydroxyurea did not alter the coumarin induced changes in cell shape. —The net RNA-synthesis per cell was slightly decreased after coumarin treatment, but the net RNA-synthesis per cell and the morphogenetic effects exerted by coumarin were not related with each other. Inhibition of m-RNA-synthesis with actinomycin D did not prevent the effects of coumarin on cell division, cell expansion, dry matter production and net protein synthesis. The same was true for inhibitors of protein synthesis, puromycin and p-fluorophenyl-alanine. The findings are in support of the view that coumarin affects already existing structures or enzymes. —Comparisons between coumarin and the uncouplers, DNP and dicoumarol, showed that the effects of coumarin were not, solely, due to uncoupling. SH-protecting agents, BAL, DTE and glutathione, did, with few exceptions, not reduce the morphogenetic effects of coumarin.     

Phylogenomic species delimitation and host‐symbiont coevolution in the fungus‐farming ant genus Sericomyrmex Mayr (Hymenoptera: Formicidae): ultraconserved elements (UCEs) resolve a recent radiation

Ants in the Neotropical genus Sericomyrmex Mayr cultivate fungi for food. Both ants and fungi are obligate, coevolved symbionts. The taxonomy of Sericomyrmex is problematic because the morphology of the worker caste is generally homogeneous across all of the species within the genus, species limits are vague, and the relationships between them are unknown. We used ultraconserved elements (UCEs) as genome‐scale markers to reconstruct evolutionary history and to infer species boundaries in Sericomyrmex. We recovered an average of ～990 UCE loci for 88 Sericomyrmex samples from across the geographical range of the genus as well as for five outgroup taxa. Using maximum likelihood and species‐tree approaches, we recovered nearly identical topologies across datasets with 50–95% matrix completeness. We identify nine species‐level lineages in Sericomyrmex, including two new species. This is less than the previously described 19 species, even accounting for two species for which we had no UCE samples, which brings the total number of Sericomyrmex species to 11. Divergence‐dating analyses recovered 4.3 Ma as the crown‐group age estimates for Sericomyrmex, indicating a recent, rapid radiation. We also sequenced mitochondrial cytochrome oxidase subunit I (COI) for 125 specimens. Resolution and support for clades in our COI phylogeny are weak, indicating that COI is not an appropriate species‐delimitation tool. However, taxa within species consistently cluster together, suggesting that COI is useful as a species identification (‘DNA barcoding’) tool. We also sequenced internal transcribed spacer (ITS) and large subunit (LSU) for 32 Sericomyrmex fungal cultivars. The fungal phylogeny confirms that Sericomyrmex fungi are generalized higher‐attine cultivars, interspersed with Trachymyrmex‐associated fungal species, indicating cultivar sharing and horizontal transfer between these two genera. Our results indicate that UCEs offer immense potential for delimiting and resolving relationships of problematic, recently diverged species.     

Multiple histone modifications in euchromatin promote heterochromatin formation by redundant mechanisms in Saccharomyces cerevisiae

Background  

Methylation of lysine 79 on histone H3 by Dot1 is required for maintenance of heterochromatin structure in yeast and humans. However, this histone modification occurs predominantly in euchromatin. Thus, Dot1 affects silencing by indirect mechanisms and does not act by the recruitment model commonly proposed for histone modifications. To better understand the role of H3K79 methylation gene silencing, we investigated the silencing function of Dot1 by genetic suppressor and enhancer analysis and examined the relationship between Dot1 and other global euchromatic histone modifiers.     

Evolutionary dynamics of the kinetochore network in eukaryotes as revealed by comparative genomics

During eukaryotic cell division, the sister chromatids of duplicated chromosomes are pulled apart by microtubules, which connect via kinetochores. The kinetochore is a multiprotein structure that links centromeres to microtubules, and that emits molecular signals in order to safeguard the equal distribution of duplicated chromosomes over daughter cells. Although microtubule‐mediated chromosome segregation is evolutionary conserved, kinetochore compositions seem to have diverged. To systematically inventory kinetochore diversity and to reconstruct its evolution, we determined orthologs of 70 kinetochore proteins in 90 phylogenetically diverse eukaryotes. The resulting ortholog sets imply that the last eukaryotic common ancestor (LECA) possessed a complex kinetochore and highlight that current‐day kinetochores differ substantially. These kinetochores diverged through gene loss, duplication, and, less frequently, invention and displacement. Various kinetochore components co‐evolved with one another, albeit in different manners. These co‐evolutionary patterns improve our understanding of kinetochore function and evolution, which we illustrated with the RZZ complex, TRIP13, the MCC, and some nuclear pore proteins. The extensive diversity of kinetochore compositions in eukaryotes poses numerous questions regarding evolutionary flexibility of essential cellular functions.     

A large-subunit mitochondrial ribosomal DNA sequence translocated to the nuclear genome of two stone crabs (Menippe)

Two DNA sequences that appear to be homologous to large-subunit mitochondrial ribosomal RNA genes have been identified in the stone crabs Menippe mercenaria and M. adina. Amplification from whole genomic DNA by polymerase chain reaction (PCR) with oligonucleotide primers based on conserved portions of large-subunit mitochondrial rRNA genes consistently amplified two products of similar length (565 and 567 bp). These products differed at 3% of their nucleotide bases, and could be distinguished by a HindIII site. Only one of these sequences (designated the A sequence) was detected by PCR in purified mitochondrial DNA. The other (designated the B sequence) hybridized to total genomic DNA at a level consistent with a nuclear genome location. It is unlikely that the type B product would have been recognized as a nuclear copy by examination of its sequence alone. This is the first report of a mitochondrial gene sequence translocated into the nuclear genome of a crustacean.      

Do Plant Cells Secrete Exosomes Derived from Multivesicular Bodies?

Multivesicular bodies (MVBs) are spherical endosomal organelles containing small vesicles formed by inward budding of the limiting membrane into the endosomal lumen. In mammalian red cells and cells of immune system, MVBs fuse with the plasma membrane in an exocytic manner, leading to release their contents including internal vesicles into the extracellular space. These released vesicles are termed exosomes. Transmission electron microscopy studies have shown that paramural vesicles situated between the plasma membrane and the cell wall occur in various cell wall-associated processes and are similar to exosomes both in location and in morphology. Our recent studies have revealed that MVBs and paramural vesicles proliferate when cell wall appositions are rapidly deposited beneath fungal penetration attempts or during plugging of plasmodesmata between hypersensitive cells and their intact neighboring cells. This indicates a potential secretion of exosome-like vesicles into the extracellular space by fusion of MVBs with the plasma membrane. This MVB-mediated secretion pathway was proposed on the basis of pioneer studies of MVBs and paramural vesicles in plants some forty years ago. Here, we recall the attention to the occurrence of MVB-mediated secretion of exosomes in plants.Key Words: cell wall, endocytosis, endosome, exocytosis, exosome, multivesicular body, paramural bodyMultivesicular bodies (MVBs) are spherical endosomal organelles containing a number of small vesicles formed by inward budding of the limiting membrane into the endosomal lumen.¹ MVBs contain endocytosed cargoes and deliver them into lysosomal/vacuolar compartments for degradation. They also incorporate newly synthesized proteins destined for lysosomal/vacuolar compartments.² In mammalian cells of hematopoietic origin, endosomal MVBs function in removal of endocytosed surface proteins in an exocytic manner. They are redirected to the plasma membrane, where they release their contents including internal vesicles into the extracellular space by membrane fusion. The released vesicles are termed exosomes.³ During reticulocyte maturation to erythrocyte, a group of surface proteins, such as the transferrin receptor, become obsolete and are discarded via MVB-mediated secretion.³ Time-course transmission electron microscopy (TEM) first revealed that colloidal gold-transferrin was internalized into MVBs via receptor-mediated endocytosis and then transferrin together with its receptor were delivered into the extracellular space via the fusion of MVBs with the plasma membrane of reticulocytes.⁴ Some other cell types of hematopoietic origin, such as activated platelets, cytotoxic T cells and antigen-presenting cells, also secrete exosomes. Exosomes thus may play a role in various physiological processes other than discarding obsolete proteins.³Our recent TEM studies provided ultrastructural evidence on the enhanced vesicle trafficking in barley leaf cells attacked by the biotrophic powdery mildew fungus. Multivesicular compartments including MVBs, intravacuolar MVBs, and paramural bodies turned out to proliferate in intact host cells during formation of cell wall appositions (papilla response), in the hypersensitive response, and during accommodation of haustoria.^5,6 MVBs proliferated in the cytoplasm of haustorium-containing epidermal cells during compatible interactions and near sites of cell wall-associated oxidative microburst either during the papilla response or during the hypersensitive response. Because MVBs in plant cells have been demonstrated to be endosomal compartments,^7–9 they may participate in internalization of nutrients from the apoplast of intact haustorium-containing epidermal cells and sequestration of damaged membranes and deleterious materials originating from the oxidative microburst.^5,6 The presence of intravacuolar MVBs with double limiting membranes (Fig. 1A) indicates an engulfment of MVBs by the tonoplast and a vacuole-mediated autophagy of MVBs.^5,6 MVBs, as prevacuolar compartments in plant cells,⁹ thus probably deliver their contents into the central vacuole via both the fusion with the tonoplast and the engulfment by the tonoplast (Fig. 2A and B). On the other hand, paramural bodies, in which small vesicles are situated between the cell wall and the plasma membrane, were associated with cell wall appositions deposited beneath fungal penetration attempts (Fig. 1B) or around hypersensitive cells including sites of plugged plasmodesmata (Fig. 1C and D).^5,6 Because paramural vesicles are similar to exosomes both in location and in morphology, we speculated that MVBs fuse with the plasma membrane in an exocytic manner to form paramural bodies.^5,6 Endocytosed cell surface materials in endosomal MVBs may be reused and delivered together with newly synthesized materials in Golgi apparatus-derived vesicles to cell wall appositions, which are deposited rapidly to prevent fungal penetration (Fig. 2A) or to contain hypersensitive cell death (Fig. 2B). MVBs thus may be driven along two distinct pathways to deliver their contents into either central vacuole or extracellular space.Open in a separate windowFigure 1Multivesicular compartments in intact cells in barley leaves attacked by the barley powdery mildew fungus. (A) An intravacuolar multivesicular body (MVB) with double limiting membranes in an intact epidermal cell (EC) adjacent to a hypersensitive epidermal cell (EC*). The arrows point to the outer limiting membrane, which is seemingly derived from the tonoplast. Note that neighboring intravacuolar vesicles (in between two arrowheads) may result from degradation of double limiting membranes of intravacuolar MVBs or may be delivered into the vacuole by MVB-fusion with the tonoplast. (B) Paramural vesicles (arrowheads) in a paramural body associated with cell wall appositions (asterisk) deposited by an intact epidermal cell. (C) A multivesicular body (MVB) in contact with a paramural body (PMB) (a nonmedian section) associated with cell wall appositions (asterisk) deposited by an intact mesophyll cell adjacent to a hypersensitive mesophyll cell. Note that cell wall appositions deposit beside an intercellular space (IS). The arrows point to the tonoplast. (D) A paramural body (PMB) associated with cell wall appositions (asterisks) blocking plasmodesmata (in between two arrowheads) at the side of an intact mesophyll cell (MC) underlying a hypersensitive epidermal cell (EC*). The arrows point to the tonoplast. CV, central vacuole; CW, cell wall; MB, microbody. Bars, 1µm.Open in a separate windowFigure 2Hypothetical diagram of delivery of endocytosed cell surface materials via MVBs into the central vacuole or the extracellular space where intact barley cells deposit cell wall appositions. (A) Deposition of cell wall appositions (asterisk) beneath powdery mildew penetration attempts. AGT, appressorial germ tube; PP, penetration peg. (B) Deposition of cell wall appositions (asterisks) against constricted plasmodesmata (PD) between a hypersensitive epidermal cell (EC) penetrated by the powdery mildew fungus and an underlying mesophyll cell (MC). H, haustorium. Arrows and numbers show pathways of vesicle trafficking. 1, Secretion of Golgi-derived vesicles containing newly synthesized materials; G, Golgi body; TGN, trans-Golgi network; 2, Endocytosis of cell surface materials from coated pits (coated open circles) via coated vesicles (coated circles) to multivesicular bodies (MVB); 3, Delivery of endocytosed materials for degradation inside the central vacuole (CV) via membrane fusion between MVBs and the tonoplast (T); small broken circles, vesicles in degradation; 4, Delivery of endocytosed materials for degradation inside the central vacuole via engulfment of MVBs by the tonoplast; large broken circles; MVB limiting membranes in degradation; 5, delivery of endocytosed materials into the extracellular space for deposition of cell wall appositions (asterisks) via membrane fusion between MVBs and the plasma membrane (PM). CW, cell wall; PMB, paramural body. PD0, 1, 2, 3 and 4 represent stages of plugging plasmodesmata. PD0, open plasmodesmata between two intact mesophyll cells (MC) subjacent to the hypersensitive epidermal cell (EC); PD1, constriction of plasmodesmata by callose (grey dots) deposition at plasmodesmal neck region; PD2, constricted plasmodesmata associated with plasmodesma-targeted secretion; PD3, further blocking of plasmodesmata by deposition of cell wall appositions; PD4, completely blocked plasmodesmata.Earlier than the discovery in animal cell systems,⁴ it was proposed in two independent papers in 1967 that the fusion of MVBs with the plasma membrane might result in the release of small vesicles into the extracellular space in fungi and in higher plants.^10,11 Several lines of evidence support the occurrence of MVB-mediated secretion of exosome-like vesicles in plants. First, vesicles of the same morphology as MVB internal vesicles have been observed in extracellular spaces or paramural spaces in various types of plant cells in various plant species by TEM.¹² An early study on endocytosis by soybean protoplasts also showed small extracellular vesicles attaching on the plasma membrane.⁸ Second, cooccurrence of MVBs and paramural vesicles has been observed in processes of cell proliferation, cell differentiation, and cell response to abiotic and biotic stress. Examples are cell plate formation,^13,14 secondary wall thickening,^15,16 cold hardness,^17,18 and deposition of cell wall appositions upon pathogen attack.^5,6,19–21 Third, identical molecular components, such as arabinogalactan proteins^22,23 and peroxidases,⁶ have been immunolocalized in both MVBs and paramural bodies. Despite these pieces of evidence, a conclusive demonstration of MVB-mediated secretion of exosomes in plants requires further exploration.The presently available experimental systems, approaches, and membrane markers may allow future demonstration of MVB-mediated secretion of exosomes in plants. Recent in vivo real-time observation and colocalization of cell surface and endosomal markers have already revealed that endosomes filled with endocytosed preexisting cell wall and plasma membrane materials are rapidly delivered to cytokinetic spaces to form cell plates in dividing tobacco, Arabidopsis, and maize cells.²⁴ Because TEM observed paramural bodies attaching to cell plates¹³ and MVBs in the vicinity of cell plates during all stages of cell plate formation,^14,25,26 MVBs and paramural bodies may participate in delivery of endocytosed building blocks to cell plates. Jiang''s and Robinson''s labs together developed a transgenic tobacco BY-2 cell line stably expressing a YFP-labeled vacuolar sorting receptor protein and antibodies against the vacuolar sorting receptor protein localized to the limiting membrane of MVBs.⁹ These tools together with live cell imaging and immunoelectron microscopy may allow visualization of MVB-fusion to the new plasma membrane, of vacuolar sorting receptors in both the limiting membrane of MVBs and the new plasma membrane, and of identical cell plate components in both internal vesicles of MVBs and paramural vesicles.In spite of obvious differences in plant and animal cytokinesis, the generation of cell plates by cell-plate-directed fusion of endosomes resembles the plugging of midbody canals by midbody-directed endosomes to separate daughter cells at the terminal phase of animal cytokinesis.²⁷ Likely, functional similarities of the fusion between endosomal MVBs and the plasma membrane to eliminate unwanted cell contents may also exist in maturation of mammalian red blood cells and plant sieve elements in the sense that the fusion of MVBs with the plasma membrane may occur during maturation of the latter.²⁸ On the other hand, although plant cells may secrete MVB-derived exosomes in defense response upon pathogen attack,^5,6 plant cell walls rule out the direct intercellular communication during the immune response mediated by exosomes in the circulation of mammals.³ In contrast, plasmodesma-directed secretion of exosomes would block the cell-to-cell communication between hypersensitive cells and their neighboring cells during hypersensitive response.⁵ Further exploration will lead us to a better understanding of similarities and differences of exosome secretion between plants and animals.