Microevolution of the mitochondrial DNA control region in the Japanese brown bear (Ursus arctos) population.

We investigated nucleotide sequences of the mitochondrial DNA control region to describe natural genetic variations and to assess the relationships between subpopulations of the brown bear Ursus arctos on Hokkaido Island, Japan. Using the polymerase chain reaction product-direct sequencing technique, partial sequences (about 930 bases) of the control region were determined for 56 brown bears sampled throughout Hokkaido Island. A sequence alignment revealed that the brown bear control region included a variable sequence on the 5' side and a repetitive region on the 3' side. Phylogenetic trees reconstructed from the 5' variable region (696-702 bases) exhibited 17 haplotypes, which were clustered into three groups (Clusters A, B, and C). The distribution of each group did not overlap with those of the others, and the three different areas were located in separate mountainous forests of Hokkaido Island. Furthermore, most of the phylogenetically close haplotypes within each group were distributed geographically close to each other. In addition, the 3' repetitive region (arrays of 10 bases) exhibited a much faster mutation rate than the 5' variable region, resulting in heteroplasmy. Such mitochondrial DNA divergence in each group could have occurred after the brown bears migrated from the continent to Hokkaido and became fixed in the different areas.     

Fusarium species—a promising tool box for industrial biotechnology

Applied Microbiology and Biotechnology - Global demand for biotechnological products has increased steadily over the years. Thus, need for optimized processes and reduced costs appear as a key...     

Acrolein-detoxifying isozymes of glutathione transferase in plants

Main conclusion

Acrolein is a lipid-derived highly reactive aldehyde, mediating oxidative signal and damage in plants. We found acrolein-scavenging glutathione transferase activity in plants and purified a low K _M isozyme from spinach.

Various environmental stressors on plants cause the generation of acrolein, a highly toxic aldehyde produced from lipid peroxides, via the promotion of the formation of reactive oxygen species, which oxidize membrane lipids. In mammals, acrolein is scavenged by glutathione transferase (GST; EC 2.5.1.18) isozymes of Alpha, Pi, and Mu classes, but plants lack these GST classes. We detected the acrolein-scavenging GST activity in four species of plants, and purified an isozyme showing this activity from spinach (Spinacia oleracea L.) leaves. The isozyme (GST-Acr), obtained after an affinity chromatography and two ion exchange chromatography steps, showed the K _M value for acrolein 93 μM, the smallest value known for acrolein-detoxifying enzymes in plants. Peptide sequence homology search revealed that GST-Acr belongs to the GST Tau, a plant-specific class. The Arabidopsis thaliana GST Tau19, which has the closest sequence similar to spinach GST-Acr, also showed a high catalytic efficiency for acrolein. These results suggest that GST plays as a scavenger for acrolein in plants.

     

The aux1 gene of the Ri plasmid is sufficient to confer auxin autotrophy in tobacco BY-2 cells

Tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells are rapidly proliferating meristematic cells that require auxin for culture in vitro. We have established several transgenic BY-2 cell lines that carry the T-DNA of Agrobacterium rhizogenes 15834, which harbors an agropine-type root-inducing (Ri) plasmid. Two of these lines, BYHR-3 and BYHR-7, were used to test the role of auxin in the proliferation of plant cells. The lines grew rapidly in Linsmaier-Skoog (LS) medium lacking auxin and other phytohormones. The TR-DNA, containing the aux1 (tryptophan monooxygenase) and aux2 (indoleacetamide hydrolase) genes, was present in the genomes of both transgenic lines, whereas the TL-DNA, containing the rolA, B, C and D genes, was present in the genome of BYHR-7 but not BYHR-3. Since the introduction of the rolABCD genes alone did not affect the auxin requirement of BY-2 cells, the aux1 and aux2 genes, but not the rolABCD genes, appear to be relevant to the auxin autotrophy of these transgenic lines. Furthermore, the overexpression of aux1 allowed BY-2 cells to grow rapidly in the absence of auxin, suggesting the existence in plant cells of an unidentified gene whose product is functionally equivalent or similar to that of aux2 of the Ri plasmid.     

Arabidopsis dynamin-related proteins DRP3A and DRP3B are functionally redundant in mitochondrial fission, but have distinct roles in peroxisomal fission

Two similar Arabidopsis dynamin-related proteins, DRP3A and DRP3B, are thought to be key factors in both mitochondrial and peroxisomal fission. However, the functional and genetic relationships between DRP3A and DRP3B have not been fully investigated. In a yeast two-hybrid assay, DRP3A and DRP3B interacted with themselves and with each other. DRP3A and DRP3B localized to mitochondria and peroxisomes, and co-localized with each other in leaf epidermal cells. In two T-DNA insertion mutants, drp3a and drp3b , the mitochondria are a little longer and fewer in number than those in the wild-type cells. In the double mutant, drp3a/drp3b , mitochondria are connected to each other, resulting in massive elongation. Overexpression of either DRP3A or DRP3B in drp3a/drp3b restored the particle shape of mitochondria, suggesting that DRP3A and DRP3B are functionally redundant in mitochondrial fission. In the case of peroxisomal fission, DRP3A and DRP3B appear to have different functions: peroxisomes in drp3a were larger and fewer in number than those in the wild type, whereas peroxisomes in drp3b were as large and as numerous as those in the wild type, and peroxisomes in drp3a/drp3b were as large and as numerous as those in drp3a . Although overexpression of DRP3A in drp3a/drp3b restored the shape and number of peroxisomes, overexpression of DRP3B did not restore the phenotypes, and often caused elongation instead. These results suggest that DRP3B and DRP3A have redundant molecular functions in mitochondrial fission, whereas DRP3B has a minor role in peroxisomal fission that is distinct from that of DRP3A.     

Peroxisomes Are Required for in Vivo Nitric Oxide Accumulation in the Cytosol following Salinity Stress of Arabidopsis Plants

Peroxisomes are unique organelles involved in multiple cellular metabolic pathways. Nitric oxide (NO) is a free radical active in many physiological functions under normal and stress conditions. Using Arabidopsis (Arabidopsis thaliana) wild type and mutants expressing green fluorescent protein through the addition of peroxisomal targeting signal 1 (PTS1), which enables peroxisomes to be visualized in vivo, this study analyzes the temporal and cell distribution of NO during the development of 3-, 5-, 8-, and 11-d-old Arabidopsis seedlings and shows that Arabidopsis peroxisomes accumulate NO in vivo. Pharmacological analyses using nitric oxide synthase (NOS) inhibitors detected the presence of putative calcium-dependent NOS activity. Furthermore, peroxins Pex12 and Pex13 appear to be involved in transporting the putative NOS protein to peroxisomes, since pex12 and pex13 mutants, which are defective in PTS1- and PTS2-dependent protein transport to peroxisomes, registered lower NO content. Additionally, we show that under salinity stress (100 mm NaCl), peroxisomes are required for NO accumulation in the cytosol, thereby participating in the generation of peroxynitrite (ONOO⁻) and in increasing protein tyrosine nitration, which is a marker of nitrosative stress.Peroxisomes are single membrane-bound organelles whose basic enzymatic constituents are catalase and H₂O₂-producing flavin oxidases as their basic enzymatic and are found in virtually all eukaryotic cell types (Corpas et al., 2001; Hayashi and Nishimura, 2006; Reumann et al., 2007; Pracharoenwattana and Smith, 2008; Palma et al., 2009). These oxidative organelles are characterized by metabolic plasticity, as their enzymatic content can vary according to the organism, cell/tissue type, and environmental conditions (Mullen et al., 2001; Hayashi and Nishimura, 2003; Corpas et al., 2009a). In higher plants, peroxisomes contain a complex battery of antioxidative enzymes, such as catalase, superoxide dismutase, the components of the ascorbate-glutathione cycle, and the NADP-dehydrogenases of the pentose-P pathway (Corpas et al., 2009a). The generation of superoxide radicals has also been reported in the matrices and membranes of peroxisomes (López-Huertas et al., 1999; del Río et al., 2006). All these findings point to the important role played by peroxisomes in the cellular metabolism of reactive oxygen species (Corpas et al., 2001, 2009a; del Río et al., 2006).Nitric oxide (NO) is a free radical involved in many physiological functions under normal and stress conditions in both animal and plant cells (Arasimowicz and Floryszak-Wieczorek, 2007; Corpas et al., 2007a, 2008; Neill et al., 2008). Unlike animal systems, knowledge of NO generation and subcellular location in plants remains largely elusive, and the data are sometimes contradictory and ambiguous (Zemojtel et al., 2006; Jasid et al., 2006; Gas et al., 2009). In previous studies, we detected l-Arg-dependent nitric oxide synthase (NOS) activity in isolated pea (Pisum sativum) leaf peroxisomes (Barroso et al., 1999). In a later study, using electron paramagnetic resonance techniques, we demonstrated the presence of NO in these types of peroxisomes (Corpas et al., 2004). However, several issues, such as whether NO is released into the cytosol and the physiological function of this free radical, remain unresolved.In this study, we provide an in vivo demonstration that Arabidopsis peroxisomes are essential for NO accumulation in the cytosol, thus participating in the generation of nitrosative stress under salinity conditions. In addition, using Arabidopsis mutants pex12 and pex13, we also suggest that these peroxins are involved in importing into peroxisomes the enzyme responsible for NO generation.     

Evaluation of the toxicity of stress-related aldehydes to photosynthesis in chloroplasts

Aldehydes produced under various environmental stresses can cause cellular injury in plants, but their toxicology in photosynthesis has been scarcely investigated. We here evaluated their effects on photosynthetic reactions in chloroplasts isolated from Spinacia oleracea L. leaves. Aldehydes that are known to stem from lipid peroxides inactivated the CO₂ photoreduction to various extents, while their corresponding alcohols and carboxylic acids did not affect photosynthesis. α,β-Unsaturated aldehydes (2-alkenals) showed greater inactivation than the saturated aliphatic aldehydes. The oxygenated short aldehydes malondialdehyde, methylglyoxal, glycolaldehyde and glyceraldehyde showed only weak toxicity to photosynthesis. Among tested 2-alkenals, 2-propenal (acrolein) was the most toxic, and then followed 4-hydroxy-(E)-2-nonenal and (E)-2-hexenal. While the CO₂-photoreduction was inactivated, envelope intactness and photosynthetic electron transport activity (H₂O → ferredoxin) were only slightly affected. In the acrolein-treated chloroplasts, the Calvin cycle enzymes phosphoribulokinase, glyceraldehyde-3-phosphate dehydrogenase, fructose-1,6-bisphophatase, sedoheptulose-1,7-bisphosphatase, aldolase, and Rubisco were irreversibly inactivated. Acrolein treatment caused a rapid drop of the glutathione pool, prior to the inactivation of photosynthesis. GSH exogenously added to chloroplasts suppressed the acrolein-induced inactivation of photosynthesis, but ascorbic acid did not show such a protective effect. Thus, lipid peroxide-derived 2-alkenals can inhibit photosynthesis by depleting GSH in chloroplasts and then inactivating multiple enzymes in the Calvin cycle.     

Quantitative PCR for Genetic Markers of Human Fecal Pollution

Assessment of health risk and fecal bacterial loads associated with human fecal pollution requires reliable host-specific analytical methods and a rapid quantification approach. We report the development of quantitative PCR assays for quantification of two recently described human-specific genetic markers targeting Bacteroidales-like cell surface-associated genes. Each assay exhibited a range of quantification from 10 to 1 × 10⁶ copies of target DNA. For each assay, internal amplification controls were developed to detect the presence or absence of amplification inhibitors. The assays predominantly detected human fecal specimens and exhibited specificity levels greater than 97% when tested against 265 fecal DNA extracts from 22 different animal species. The abundance of each human-specific genetic marker in primary effluent wastewater samples collected from 20 geographically distinct locations was measured and compared to quantities estimated by real-time PCR assays specific for rRNA gene sequences from total Bacteroidales and enterococcal fecal microorganisms. Assay performances combined with the prevalence of DNA targets in sewage samples provide experimental evidence supporting the potential application of these quantitative methods for monitoring fecal pollution in ambient environmental waters.Waterborne diseases that originate from human fecal pollution remain a significant public health issue. As a result, a large number of methods have been developed to detect and quantify human fecal pollution (10, 12, 18, 20). The majority of these methods are based on real-time quantitative PCR (qPCR) assays designed to estimate the concentrations of 16S rRNA gene sequences from various subpopulations within the order Bacteroidales. This bacterial order constitutes a large proportion of the normal gut microbiota of most animals, including humans (3, 15, 27). Bacterial 16S rRNA genes are useful as markers because they have relatively low mutation rates (7) and are typically present in multiple operons, increasing template DNA levels available for detection (2, 11, 17, 29). While several studies have demonstrated the value of Bacteroides 16S rRNA gene-based qPCR assays, currently available assays cannot discriminate between several animal sources closely associated with humans, including cats, dogs, and/or swine (10, 12, 18, 20). Alternative qPCR assays targeting genes directly involved in host-specific interactions may be capable of increased discrimination of fecal pollution sources (22, 23) and are needed to complement existing qPCR-based approaches used to identify sources of human fecal pollution.A recent metagenomic survey of a human fecal bacterial community using genome fragment enrichment has led to the identification of hundreds of candidate human fecal bacterium-specific DNA sequences (23). PCR assays targeting two gene sequences encoding a hypothetical protein potentially involved in remodeling of bacterial surface polysaccharides and lipopolysaccharides (assay 19) and a putative RNA polymerase extracytoplasmic function sigma factor (assay 22) from Bacteroidales-like microorganisms exhibited a high level of specificity (100%) for human fecal material (23). However, it remained to be determined whether these reported chromosomal DNA sequences are abundant and uniform enough within human populations to be detected once diluted in the environment. On the basis of these considerations, the next steps toward the application of these gene sequences for water quality monitoring applications were to design qPCR assays for their detection and then to use these assays to evaluate the overall abundance and distribution of these sequences in human populations relative to those of rRNA gene sequences from different currently recognized fecal indicator bacterial groups.Here, we report the development of two qPCR assays for quantification of the human-specific DNA sequences targeted by previously reported PCR assays 19 and 22 (23). Method performance characteristics, including specificity, range of quantification (ROQ), limit of quantification, amplification efficiency, and analytical precision, were defined for each assay. An internal amplification control (IAC) was designed to monitor for the presence of inhibitors commonly associated with environmental sampling that can confound DNA target copy number estimations. Finally, the abundance of each DNA target in primary effluent wastewater samples representative of 20 geographically distinct human populations was measured by qPCR analysis. In addition, the abundances of these human-specific DNA genes in wastewater were compared to those of rRNA genes of Bacteroidales and enterococci, two general fecal indicator bacterial groups that have been widely used for water quality testing.