Effect of culture media components on accumulation of glutaminyl endopeptidase in culture liquid of Bacillus intermedius 3-19

The effect of nutrients and growth conditions on the accumulation of glutamyl endopeptidase in the culture liquid of Bacillus intermedius 3-19 was studied. Glucose and other readily metabolizable carbon sources were found to suppress the production of the enzyme, while inorganic phosphate and ammonium cations enhanced it. Protein substrates, such as casein, gelatin, and hemoglobin, did not affect enzyme production. Some bivalent cations (Ca2+, Mg2+, Co2+) increased the production of glutamyl endopeptidase, but others (Zn2+, Fe2+, Cu2+) acted in the opposite way. The rate of enzyme accumulation in the culture liquid increased as the growth rate of the bacterium decreased, so that the maximum enzyme activity was observed in the stationary growth phase. Based on the results of this investigation, an optimal medium for the maximum production of glutamyl endopeptidase by B. intermedius 3-19 was elaborated.     

Biological significance of the Gornoslinkinskaya Riverbed Depression in the Irtysh

Distribution of fish in the Gornoslinkinskaya Riverbed Depression in the Irtysh is considered in relation to the time of day and to the season. Fish aggregations are characterized quantitatively and by their size and species composition. The major part of the fish population of the depression consists of fish juveniles. The biological significance of the riverbed depression is polyfunctional. The universal traits of the ecosystems of large river basins and mechanisms of their occupation by fish are discussed.     

A C-terminal Myb extension domain defines a novel family of double-strand telomeric DNA-binding proteins in Arabidopsis

Little is known about the protein composition of plant telomeres. We queried the Arabidopsis thaliana genome data base in search of genes with similarity to the human telomere proteins hTRF1 and hTRF2. hTRF1/hTRF2 are distinguished by the presence of a single Myb-like domain in their C terminus that is required for telomeric DNA binding in vitro. Twelve Arabidopsis genes fitting this criterion, dubbed TRF-like (TRFL), fell into two distinct gene families. Notably, TRFL family 1 possessed a highly conserved region C-terminal to the Myb domain called Myb-extension (Myb-ext) that is absent in TRFL family 2 and hTRF1/hTRF2. Immunoprecipitation experiments revealed that recombinant proteins from TRFL family 1, but not those from family 2, formed homodimers and heterodimers in vitro. DNA binding studies with isolated C-terminal fragments from TRFL family 1 proteins, but not family 2, showed specific binding to double-stranded plant telomeric DNA in vitro. Removal of the Myb-ext domain from TRFL1, a family 1 member, abolished DNA binding. However, when the Myb-ext domain was introduced into the corresponding region in TRFL3, a family 2 member, telomeric DNA binding was observed. Thus, Myb-ext is required for binding plant telomeric DNA and defines a novel class of proteins in Arabidopsis.     

POT1 proteins in green algae and land plants: DNA-binding properties and evidence of co-evolution with telomeric DNA

Telomeric DNA terminates with a single-stranded 3′ G-overhang that in vertebrates and fission yeast is bound by POT1 (Protection Of Telomeres). However, no in vitro telomeric DNA binding is associated with Arabidopsis POT1 paralogs. To further investigate POT1–DNA interaction in plants, we cloned POT1 genes from 11 plant species representing major branches of plant kingdom. Telomeric DNA binding was associated with POT1 proteins from the green alga Ostreococcus lucimarinus and two flowering plants, maize and Asparagus. Site-directed mutagenesis revealed that several residues critical for telomeric DNA recognition in vertebrates are functionally conserved in plant POT1 proteins. However, the plant proteins varied in their minimal DNA-binding sites and nucleotide recognition properties. Green alga POT1 exhibited a strong preference for the canonical plant telomere repeat sequence TTTAGGG with no detectable binding to hexanucleotide telomere repeat TTAGGG found in vertebrates and some plants, including Asparagus. In contrast, POT1 proteins from maize and Asparagus bound TTAGGG repeats with only slightly reduced affinity relative to the TTTAGGG sequence. We conclude that the nucleic acid binding site in plant POT1 proteins is evolving rapidly, and that the recent acquisition of TTAGGG telomere repeats in Asparagus appears to have co-evolved with changes in POT1 DNA sequence recognition.     

POT1-independent single-strand telomeric DNA binding activities in Brassicaceae

Telomeres define the ends of linear eukaryotic chromosomes and are required for genome maintenance and continued cell proliferation. The extreme ends of telomeres terminate in a single-strand protrusion, termed the G-overhang, which, in vertebrates and fission yeast, is bound by evolutionarily conserved members of the POT1 (protection of telomeres) protein family. Unlike most other model organisms, the flowering plant Arabidopsis thaliana encodes two divergent POT1-like proteins. Here we show that the single-strand telomeric DNA binding activity present in A. thaliana nuclear extracts is not dependent on POT1a or POT1b proteins. Furthermore, in contrast to POT1 proteins from yeast and vertebrates, recombinant POT1a and POT1b proteins from A. thaliana , and from two additional Brassicaceae species, Arabidopsis lyrata and Brassica oleracea (cauliflower), fail to bind single-strand telomeric DNA in vitro under the conditions tested. Finally, although we detected four single-strand telomeric DNA binding activities in nuclear extracts from B. oleracea , partial purification and DNA cross-linking analysis of these complexes identified proteins that are smaller than the predicted sizes of BoPOT1a or BoPOT1b. Taken together, these data suggest that POT1 proteins are not the major single-strand telomeric DNA binding activities in A. thaliana and its close relatives, underscoring the remarkable functional divergence of POT1 proteins from plants and other eukaryotes.     

Promises and Challenges of Eco-Physiological Genomics in the Field: Tests of Drought Responses in Switchgrass

Identifying the physiological and genetic basis of stress tolerance in plants has proven to be critical to understanding adaptation in both agricultural and natural systems. However, many discoveries were initially made in the controlled conditions of greenhouses or laboratories, not in the field. To test the comparability of drought responses across field and greenhouse environments, we undertook three independent experiments using the switchgrass reference genotype Alamo AP13. We analyzed physiological and gene expression variation across four locations, two sampling times, and three years. Relatively similar physiological responses and expression coefficients of variation across experiments masked highly dissimilar gene expression responses to drought. Critically, a drought experiment utilizing small pots in the greenhouse elicited nearly identical physiological changes as an experiment conducted in the field, but an order of magnitude more differentially expressed genes. However, we were able to define a suite of several hundred genes that were differentially expressed across all experiments. This list was strongly enriched in photosynthesis, water status, and reactive oxygen species responsive genes. The strong across-experiment correlations between physiological plasticity—but not differential gene expression—highlight the complex and diverse genetic mechanisms that can produce phenotypically similar responses to various soil water deficits.Crop productivity and wild plant distributions are governed by the availability of soil moisture (Axelrod, 1972; Boyer, 1982; Ciais et al., 2005). The impact of drought and soil water deficit in agriculture is estimated to be the largest abiotic determinant of yield (Boyer, 1982; Araus et al., 2002), while drought is also considered a primary cause of speciation and adaptation in nature (Stebbins, 1952). Dehydration avoidance and other drought adaptive strategies permit plants to survive or maintain growth during periodic droughts (Blum, 1996; Chaves et al., 2003; Chaves and Oliveira, 2004). Specifically, phenotypic plasticity of stomatal conductance, water foraging, and growth traits (among many others) may effectively maintain homeostasis of leaf water potential despite soil water deficits.Leaf water potential is a bellwether of the physiological impact of water deficit (Jones, 2007). Under drought, decreasing water availability results in reduced leaf water potentials and a sequence of physiological responses including reduced photosynthesis, growth rate, and ultimately, fitness (Taiz and Zeiger, 2014). Plants therefore seek to maintain homeostasis of leaf water potential, with the highest (least negative) values supporting the most efficient functioning of photosynthesis and other metabolic processes in most species (Lawlor and Fock, 1978; Turner and Begg, 1981; Kramer and Boyer, 1995; Cornic and Massacci, 1996; Jones, 2007). Plants that exhibit dehydration avoidance strategies compensate for soil water deficit through phenotypic plasticity of gene expression (Verslues et al., 2006; DesMarais and Juenger, 2010; DesMarais et al., 2013; Lovell et al., 2015) and downstream physiological phenotypes (Levitt, 1980), among others.To understand plant stress responses, it is critical to determine the physiological and genetic underpinnings of drought adaptation in both field and laboratory conditions (Travers et al., 2007; Gaudin et al., 2013). A common finding among such studies is that physiological and gene expression responses to drought vary considerably depending on the severity and temporal dynamics of drying soil (Chaves et al., 2003; Barker et al., 2005; Malmberg et al., 2005; Mittler, 2006; Mishra et al., 2012). Natural soil moisture variation, which has shaped adaptive responses to drought in wild populations, is not necessarily recapitulated by controlled (often, “shock”) laboratory experiments. For example, single abiotic stresses rarely occur in isolation in the field (Mittler, 2006). Instead, wild and crop plants respond to the combination of diverse stressors such as drought, heat, and salinity, simultaneously and at both molecular (e.g. Rizhsky et al., 2002; Rizhsky et al., 2004; Suzuki et al., 2005) and physiological (e.g. Heyne and Brunson, 1940; Craufurd and Peacock, 1993; Machado and Paulsen, 2001) levels. Therefore, inquiries into evolved plant stress responses are perhaps best served by experimental conditions that emulate selective agents in the field. Given that the extent and severity of stress causes qualitatively different physiological responses, it is not surprising that several studies have found relatively weak genetic correlations between laboratory phenotypes and those collected in the field (e.g. Weinig et al., 2002; Malmberg et al., 2005; Anderson et al., 2011; Mishra et al., 2012).Soil properties and biota can also affect plant growth and physiology (Meisner et al., 2013; Schweitzer et al., 2014), which may be exacerbated by contrasts between growth in potting mix or in native soil (Rowe et al., 2007; Heinze et al., 2016). The observation that field-grown plants have different root systems and greater total water storage than those in greenhouse pots is of particular importance to water relations (Poorter et al., 2012a). Short-term drought stress in the field may be buffered by access to larger volumes of soil and more complex root-soil-water dynamics, conditions poorly represented in most controlled settings.The field of experimental design has been fundamentally shaped by a central problem of biology: that it is notoriously difficult to control environmental factors in the field (Jones, 2013). A classic solution is to increase biological replication, but this is generally not feasible with costly and time-sensitive physiological and genetic assays (Poorter et al., 2012b; Marchand et al., 2013). Despite these difficulties, understanding the effects of drought in field conditions is necessary because it is in these settings that yield is impacted and selection is acting to shape adaptive responses to stress. Here, we determine how the interplay between drought severity, planting condition (e.g. field, potted, greenhouse) and sampling timing impacts physiological and genomic responses to drought in the C₄ perennial grass, Panicum virgatum (switchgrass). To accomplish this, we used observations collected from clonally replicated individuals of the “AP13” switchgrass genotype (derived from the Alamo cultivar), which is the genome reference for this important biofuel crop and dominant member of mesic tall grass prairie ecosystems. The Alamo cultivar is a southern lowland accession that has high vigor and performance across a variety of climatic conditions. Replicates were grown in three separate soil moisture manipulation experiments with distinct rooting environments: in medium sized pots in a greenhouse, in large containers in a field setting, and in native soil under rainout shelters. In all three of these experiments, we collected leaf-level physiological and whole-genome gene expression data from droughted and control plants.Combined, the three experiments represent contrasts in drought experimental manipulations (i.e. the extent, timing, and duration of drought), plant characteristics (i.e. age, maturity, and size), and broadly fit with the concepts of best practice for physiological analysis of drought responses (Poorter et al., 2012b). Contrasting these experimental design considerations allows us to address how edaphic and climactic conditions impact links between gene expression and physiological phenotypic plasticity. Specifically, we assessed three fundamental questions pertaining to physiological genomics in the field: (1) How consistent is phenotypic plasticity to drought across experiments? (2) Which soil moisture deficit responses vary across sites, years, and timing of sampling? (3) How does plasticity of physiological and gene expression phenotypes covary within and across experiments? To assess these questions, we tested how leaf physiology and whole-genome gene expression responded to the effects of drought treatments, leaf water potential, and sampling time (midday and predawn). These analyses permitted inference of the number, relative effect size, and identity of differentially expressed (plastic) genes. Overall, our results suggested that differences in leaf water potential and diurnal patterns were the major drivers of gene expression variation. Furthermore, we observed consistent physiological plasticity across greenhouse dry-down and field precipitation manipulation experiments, but extreme variability in the number of differentially expressed genes.     

Structural characteristics and catalytic mechanism of <Emphasis Type="Italic">Bacillus</Emphasis> β-propeller phytases

ß-Propeller phytases of Bacillus are unique highly conservative and highly specific enzymes capable of cleaving insoluble phytate compounds. In this review, we analyzed data on the properties of these enzymes, their differences from other phytases, and their unique spatial structures and substrate specificities. We considered influences of different factors on the catalytic activity and thermostability of these enzymes. There are few data on the hydrolysis mechanism of these enzymes, which makes it difficult to analyze their mechanism of action and their final products. We analyzed the available data on hydrolysis by ß-propeller phytases of calcium complexes with myo-inositol hexakisphosphate.     

The Arabidopsis Pot1 and Pot2 proteins function in telomere length homeostasis and chromosome end protection

Pot1 (protection of telomeres 1) is a single-stranded telomere binding protein that is essential for chromosome end protection and telomere length homeostasis. Arabidopsis encodes two Pot1-like proteins, dubbed AtPot1 and AtPot2. Here we show that telomeres in transgenic plants expressing a truncated AtPot1 allele lacking the N-terminal oligonucleotide/oligosaccharide binding fold (P1DeltaN) are 1 to 1.5 kb shorter than in the wild type, suggesting that AtPot1 contributes to the positive regulation of telomere length control. In contrast, telomere length is unperturbed in plants expressing the analogous region of AtPot2. A strikingly different phenotype is observed in plants overexpressing the AtPot2 N terminus (P2DeltaC) but not the corresponding region in AtPot1. Although bulk telomeres in P2DeltaC mutants are 1 to 2 kb shorter than in the wild type, these plants resemble late-generation telomerase-deficient mutants with severe growth defects, sterility, and massive genome instability, including bridged chromosomes and aneuploidy. The genome instability associated with P2DeltaC mutants implies that AtPot2 contributes to chromosome end protection. Thus, Arabidopsis has evolved two Pot genes that function differently in telomere biology. These findings provide unanticipated information about the evolution of single-stranded telomere binding proteins.     

Effects of natural and artificial defoliations on the content and composition of extractive substances in birch leaves

Qualitative and quantitative compositions of extracts of birch (Betula pendula Roth.) leaves after natural and artificial defoliations were studied. Composition of the fraction of total lipids was determined. Overall, 11 fatty acids were identified. Differences between the fatty acid compositions of total lipids in the trees subjected to defoliation, consisting in the increase in quantities of short-chain saturated fatty acids and trienoic acids, were detected. Nine individual compounds--six flavones, two flavanones, and one flavanonol--were isolated from the flavonoid fraction by column chromatography. It was found that the total content of extractive substances in birch leaves as well as amounts of free sterols, triterpene compounds, and flavones decreased 1 year after an artificial defoliation and 1 month after depredation of 75% of birch stands by gypsy moth. On the contrary, the contents of flavanones and flavanonol increased. The assay method proposed may be used for studying the compositions of plant extracts.