Barley hordothionin accumulates in transgenic oat seeds and purified protein retains anti-fungal properties in vitro

Summary Transgenic anti-fungal gene expression in heterologous species provides a means to test resistance protein combinations across species barriers. This is the first report of transgenic anti-fungal seed storage protein accumulation in oat seed. An anti-fungal barley (Hordeum vulgare L.) hordothionin (Hthl) gene was genetically engineered into oat (Avena sativa L.) to determine the effect of hordothionin on pathogen resistance. The transgene was expressed in both leaf and seed tissue, with transgenic protein accumulation occurring only in the seed. Transgenic oat line HTH-Av5 expressed c. 94 μg HTH/g seed, 19% of native barley seed levels. The anti-fungal activities of HTH fractions from barley cv. Morex and oat (transgenic and control) were tested in an in vitro growth assay against an important small grain pathogen. Fusarium graminearum. The partially purified HTH fractions from control oat seeds did not inhibit fungal growth, while HPLC-purified HTH positive control, as well as partially purified barley and transgenic oat HTH inhibited growth similarly over a range of concentrations. These results indicate hordothionin can be expressed in a heterologous cereal species and still maintain its anti-fungal properties. Future studies with HTH targeted to additional tissues are planned to test for increased fungal resistance. The University of Wisconsin and the USDA neither guarantee nor warrant the standard of the products named herein, and the use of the name by University of Wisconsin or USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Hierarchical classification of switchgrass genotypes using SSR and chloroplast sequences: ecotypes,ploidies, gene pools,and cultivars

Switchgrass (Panicum virgatum L.) is an important crop for bioenergy feedstock development. Switchgrass has two main ecotypes: the lowland ecotype being exclusively tetraploid (2n = 4x = 36) and the upland ecotype being mainly tetraploid and octaploid (2n = 8x = 72). Because there is a significant difference in ploidy, morphology, growth pattern, and zone of adaptation between and within the upland and lowland ecotypes, it is important to discriminate switchgrass plants belonging to different genetic pools. We used 55 simple sequence repeats (SSR) loci and six chloroplast sequences to identify patterns of variation between and within 18 switchgrass cultivars representing seven lowland and 11 upland cultivars from different geographic regions and of varying ploidy levels. We report consistent discrimination of switchgrass cultivars into ecotype membership and demonstrate unambiguous molecular differentiation among switchgrass ploidy levels using genetic markers. Also, SSR and chloroplast markers identified genetic pools related to the geographic origin of the 18 cultivars with respect to ecotype, ploidy, and geographical, and cultivar sources. SSR loci were highly informative for cultivar fingerprinting and to classify plants of unknown origin. This classification system is the first step toward developing switchgrass complementary gene pools that can be expected to provide a significant heterotic increase in biomass yield.     

Analysis of cardiac signals using spatial filling index and time-frequency domain

Background  

Analysis of heart rate variation (HRV) has become a popular noninvasive tool for assessing the activities of the autonomic nervous system (ANS). HRV analysis is based on the concept that fast fluctuations may specifically reflect changes of sympathetic and vagal activity. It shows that the structure generating the signal is not simply linear, but also involves nonlinear contributions. These signals are essentially non-stationary; may contain indicators of current disease, or even warnings about impending diseases. The indicators may be present at all times or may occur at random in the time scale. However, to study and pinpoint abnormalities in voluminous data collected over several hours is strenuous and time consuming.     

Symmetric cyanine dyes for detecting nucleic acids

A novel approach to the design of sensitive fluorescent probes for nucleic acids detection is proposed. Suitable modifications of tri- and pentamethine cyanine dyes in the polymethine chain and/or in the heterocyclic residues can result in a significant decrease in unbound dye fluorescence intensity and an increase in dye emission intensity in the presence of DNA compared to the unsubstituted dye. The sharp enhancement in the fluorescence intensity upon dye interaction with double-stranded DNA permits the application of the modified tri- and pentamethine dyes as fluorescent probes in double-stranded DNA detection in homogeneous assays.     

The ecological integrity of the Lower Olifants River,Limpopo province,South Africa: 2009–2015 – Part A: Olifants River main stem

The major rivers of the South African ‘Lowveld’ (low-latitude savanna) suffer numerous impacts from upstream economic activities. Whereas monitoring these rivers is required to detect biodiversity losses, record pollution events and devise mitigation strategies, current monitoring programmes are inadequate. In 2009, the South African Earth Observation Network initiated an intensive long-term research programme on the Lowveld reaches of the Olifants River. Physico-chemical parameters, aquatic macroinvertebrates and fish abundances were recorded at four Lowveld sites in the Olifants River. We review six years of this programme. The results suggest deterioration in the ecological condition of the Olifants River with no discernible improvement through protected areas. Trends could not be detected. The parameters measured, sampling methods and/or sampling frequency might be responsible for the limited trends observed, or alternatively the results simply reflect stable conditions despite on-going pollution. Real time monitoring and an expansion in the parameters monitored would add value to the monitoring programme.     

Spatial and temporal divergence of expression in duplicated barley germin-like protein-encoding genes

Subfunctionalization is the process by which a pair of duplicated genes, or paralogs, experiences a reduction of individual expression patterns or function while still reproducing the complete expression pattern and function of the ancestral gene. Two germin-like protein (GLP)-encoding genes, GerB and GerF, are paralogs that belong to a small gene family in barley (Hordeum vulgare). Both genes share high nucleotide sequence similarity in coding and noncoding regions and encode identical apoplastic proteins. The use of RNA gel blots, coupled with single-stranded conformation polymorphism (SSCP) analysis of RT-PCR products, elucidated the developmental and tissue-specific expression patterns of each gene. Individual expression patterns provided evidence of both overlapping redundancy and early subfunctionalization. GerB is predominantly expressed in developing shoots, while GerF is predominantly expressed in seedling roots, developing spikes, and pericarp/testa. GerF promoter deletion studies located a region (-356/-97) responsible for high promoter activity and showed the ability of GerB and GerF upstream regions to drive gfp expression in coleoptiles, epicarps, and lemma/palea of developing spikes. The observed expression patterns are consistent with proposed roles in plant development and defense mechanisms for this gene family. These roles may explain why redundancy has been selectively maintained in this duplicate gene pair.     

A high-throughput core sampling device for the evaluation of maize stalk composition

Background

A major challenge in the identification and development of superior feedstocks for the production of second generation biofuels is the rapid assessment of biomass composition in a large number of samples. Currently, highly accurate and precise robotic analysis systems are available for the evaluation of biomass composition, on a large number of samples, with a variety of pretreatments. However, the lack of an inexpensive and high-throughput process for large scale sampling of biomass resources is still an important limiting factor. Our goal was to develop a simple mechanical maize stalk core sampling device that can be utilized to collect uniform samples of a dimension compatible with robotic processing and analysis, while allowing the collection of hundreds to thousands of samples per day.

Results

We have developed a core sampling device (CSD) to collect maize stalk samples compatible with robotic processing and analysis. The CSD facilitates the collection of thousands of uniform tissue cores consistent with high-throughput analysis required for breeding, genetics, and production studies. With a single CSD operated by one person with minimal training, more than 1,000 biomass samples were obtained in an eight-hour period. One of the main advantages of using cores is the high level of homogeneity of the samples obtained and the minimal opportunity for sample contamination. In addition, the samples obtained with the CSD can be placed directly into a bath of ice, dry ice, or liquid nitrogen maintaining the composition of the biomass sample for relatively long periods of time.

Conclusions

The CSD has been demonstrated to successfully produce homogeneous stalk core samples in a repeatable manner with a throughput substantially superior to the currently available sampling methods. Given the variety of maize developmental stages and the diversity of stalk diameter evaluated, it is expected that the CSD will have utility for other bioenergy crops as well.     

Diabetogenic diet-induced insulin resistance associates with lipid droplet proteins and adipose tissue secretome,but not with sexual dimorphic adipose tissue fat accumulation in Wistar rats

The role of sexual dimorphic adipose tissue fat accumulation in the development of insulin resistance is well known. However, whether vitamin A status and/or its metabolic pathway display any sex- or depot (visceral/subcutaneous)-specific pattern and have a role in sexual dimorphic adipose tissue development and insulin resistance are not completely understood. Therefore, to assess this, 5 weeks old Wistar male and female rats of eight from each sex were provided either control or diabetogenic (high fat, high sucrose) diet for 26 weeks. At the end, consumption of diabetogenic diet increased the visceral fat depots (p < 0.001) in the males and subcutaneous depot (p < 0.05) in the female rats, compared to their sex-matched controls. On the other hand, it caused adipocyte hypertrophy (p < 0.05) of visceral depot (retroperitoneal) in the females and subcutaneous depot of the male rats. Although vitamin A levels displayed sex- and depot-specific increase due to the consumption of diabetogenic diet, the expression of most of its metabolic pathway genes in adipose depots remained unaltered. However, the mRNA levels of some of lipid droplet proteins (perilipins) and adipose tissue secretory proteins (interleukins, lipocalin-2) did display sexual dimorphism. Nonetheless, the long-term feeding of diabetogenic diet impaired the insulin sensitivity, thus affected glucose clearance rate and muscle glucose-uptake in both the sexes of rats. In conclusion, the chronic consumption of diabetogenic diet caused insulin resistance in the male and female rats, but did not corroborate with sexual dimorphic adipose tissue fat accumulation or its vitamin A status.