Production of high levels of poly‐3‐hydroxybutyrate in plastids of Camelina sativa seeds

Poly‐3‐hydroxybutyrate (PHB) production in plastids of Camelina sativa seeds was investigated by comparing levels of polymer produced upon transformation of plants with five different binary vectors containing combinations of five seed‐specific promoters for expression of transgenes. Genes encoding PHB biosynthetic enzymes were modified at the N‐terminus to encode a plastid targeting signal. PHB levels of up to 15% of the mature seed weight were measured in single sacrificed T₁ seeds with a genetic construct containing the oleosin and glycinin promoters. A more detailed analysis of the PHB production potential of two of the best performing binary vectors in a Camelina line bred for larger seed size yielded lines containing up to 15% polymer in mature T₂ seeds. Transmission electron microscopy showed the presence of distinct granules of PHB in the seeds. PHB production had varying effects on germination, emergence and survival of seedlings. Once true leaves formed, plants grew normally and were able to set seeds. PHB synthesis lowered the total oil but not the protein content of engineered seeds. A change in the oil fatty acid profile was also observed. High molecular weight polymer was produced with weight‐averaged molecular weights varying between 600 000 and 1 500 000, depending on the line. Select lines were advanced to later generations yielding a line with 13.7% PHB in T₄ seeds. The levels of polymer produced in this study are the highest reported to date in a seed and are an important step forward for commercializing an oilseed‐based platform for PHB production.     

What's in a name; Genetic structure in Solanum section Petota studied using population-genetic tools

Background  

The taxonomy and systematic relationships among species of Solanum section Petota are complicated and the section seems overclassified. Many of the presumed (sub)species from South America are very similar and they are able to exchange genetic material. We applied a population genetic approach to evaluate support for subgroups within this material, using AFLP data. Our approach is based on the following assumptions: (i) accessions that may exchange genetic material can be analyzed as if they are part of one gene pool, and (ii) genetic differentiation among species is expected to be higher than within species.     

Synergizing metabolic flux analysis and nucleotide sugar metabolism to understand the control of glycosylation of recombinant protein in CHO cells

Background

The photorespiratory nitrogen cycle in C₃ plants involves an extensive diversion of carbon and nitrogen away from the direct pathways of assimilation. The liberated ammonia is re-assimilated, but up to 25% of the carbon may be released into the atmosphere as CO₂. Because of the loss of CO₂ and high energy costs, there has been considerable interest in attempts to decrease the flux through the cycle in C₃ plants. Transgenic tobacco plants were generated that contained the genes gcl and hyi from E. coli encoding glyoxylate carboligase (EC 4.1.1.47) and hydroxypyruvate isomerase (EC 5.3.1.22) respectively, targeted to the peroxisomes. It was presumed that the two enzymes could work together and compete with the aminotransferases that convert glyoxylate to glycine, thus avoiding ammonia production in the photorespiratory nitrogen cycle.

Results

When grown in ambient air, but not in elevated CO₂, the transgenic tobacco lines had a distinctive phenotype of necrotic lesions on the leaves. Three of the six lines chosen for a detailed study contained single copies of the gcl gene, two contained single copies of both the gcl and hyi genes and one line contained multiple copies of both gcl and hyi genes. The gcl protein was detected in the five transgenic lines containing single copies of the gcl gene but hyi protein was not detected in any of the transgenic lines. The content of soluble amino acids including glycine and serine, was generally increased in the transgenic lines growing in air, when compared to the wild type. The content of soluble sugars, glucose, fructose and sucrose in the shoot was decreased in transgenic lines growing in air, consistent with decreased carbon assimilation.

Conclusions

Tobacco plants have been generated that produce bacterial glyoxylate carboligase but not hydroxypyruvate isomerase. The transgenic plants exhibit a stress response when exposed to air, suggesting that some glyoxylate is diverted away from conversion to glycine in a deleterious short-circuit of the photorespiratory nitrogen cycle. This diversion in metabolism gave rise to increased concentrations of amino acids, in particular glutamine and asparagine in the leaves and a decrease of soluble sugars.     

Scats and den contents as indicators of the diet of stoats (Mustela erminea) in the Tasman Valley,South Canterbury,New Zealand

Stoats are significant predators of native fauna in New Zealand. They occur in many habitat types and consume a wide range of prey. The diet of stoats in the Tasman River, South Canterbury, was studied by analysis of scats and den contents. Analysis of 206 scats showed that stoats ate mainly lagomorphs, birds and invertebrates. Minor components included mice, lizards, fish and hedgehogs. Stoats ate more birds in spring than in autumn, and female stoats ate more invertebrates than did males. The contents of 219 dens collected in the same area at the same time provided further information. Birds and lagomorphs occurred at high frequency in dens, and other components were minor. Remains in dens were larger than in scats and allowed identification of many more prey items to species level. Den contents revealed a potentially substantial impact of stoats on threatened shorebirds locally; this impact was not detected by analysis of scats.     

Closed-loop seizure control with very high frequency electrical stimulation at seizure onset in the GAERS model of absence epilepsy

A closed-loop system for the automated detection and control of epileptic seizures was created and tested in three Genetic Absence Epilepsy Rats from Strasbourg (GAERS) rats. In this preliminary study, a set of four EEG features were used to detect seizures and three different electrical stimulation strategies (standard (130 Hz), very high (500 Hz) and ultra high (1000 Hz)) were delivered to terminate seizures. Seizure durations were significantly shorter with all three stimulation strategies when compared to non-stimulated (control) seizures. We used mean seizure duration of epileptiform discharges persisting beyond the end of electrical stimulation as a measure of stimulus efficacy. When compared to the duration of seizures stimulated in the standard approach (7.0 s ± 10.1), both very high and ultra high frequency stimulation strategies were more effective at shortening seizure durations (1.3 ± 2.2 s and 3.5 ± 6.4 s respectively). Further studies are warranted to further understand the mechanisms by which this therapeutic effect may be conveyed, and which of the novel aspects of the very high and ultra high frequency stimulation strategies may have contributed to the improvement in seizure abatement performance when compared to standard electrical stimulation approaches.     

Leopard frog priorities in choosing between prey at different locations

Frogs are able to respond to a prey stimulus throughout their 360° ground-level visual field as well as in the superior visual field. We compared the likelihood of frogs choosing between a more nasally located, ground-level prey versus a more temporally located ground-level prey, when the prey at the nasal location is further away from the frog. Two crickets were presented simultaneously at 9 pairs of angles that included both crickets in the binocular visual field, both crickets in the monocular visual field, or one cricket in the binocular field and one in the monocular field. Frogs chose the more nasally located prey at least 71% of the time when the more temporal prey was in the monocular field; and 64% of the time when both prey were in the binocular field. Frogs tended to choose the more nasally located prey, even though it takes the frog longer to reach the prey. In addition, when given a choice between a prey located at ground level versus a prey located in the superior field, frogs tend to choose the prey at ground-level. These results suggest that there is a neural mechanism that biases frogs’ responses to prey stimuli.     

Mechanism of the antioxidant to pro-oxidant switch in the behavior of dehydroascorbate during LDL oxidation by copper(II) ions

Oxidised low density lipoprotein (LDL) may be involved in the pathogenesis of atherosclerosis. We have therefore investigated the mechanisms underlying the antioxidant/pro-oxidant behavior of dehydroascorbate, the oxidation product of ascorbic acid, toward LDL incubated with Cu(2+) ions. By monitoring lipid peroxidation through the formation of conjugated dienes and lipid hydroperoxides, we show that the pro-oxidant activity of dehydroascorbate is critically dependent on the presence of lipid hydroperoxides, which accumulate during the early stages of oxidation. Using electron paramagnetic resonance spectroscopy, we show that dehydroascorbate amplifies the generation of alkoxyl radicals during the interaction of copper ions with the model alkyl hydroperoxide, tert-butylhydroperoxide. Under continuous-flow conditions, a prominent doublet signal was detected, which we attribute to both the erythroascorbate and ascorbate free radicals. On this basis, we propose that the pro-oxidant activity of dehydroascorbate toward LDL is due to its known spontaneous interconversion to erythroascorbate and ascorbate, which reduce Cu(2+) to Cu(+) and thereby promote the decomposition of lipid hydroperoxides. Various mechanisms, including copper chelation and Cu(+) oxidation, are suggested to underlie the antioxidant behavior of dehydroascorbate in LDL that is essentially free of lipid hydroperoxides.