Nitrogen use efficiencies of spring barley grown under varying nitrogen conditions in the field and growth chamber

Background and Aims

Nitrogen-use efficiency (NUE) of cereals needs to be improved by nitrogen (N) management, traditional plant breeding methods and/or biotechnology, while maintaining or, optimally, increasing crop yields. The aims of this study were to compare spring-barley genotypes grown on different nitrogen levels in field and growth-chamber conditions to determine the effects on N uptake (NUpE) and N utilization efficiency (NUtE) and ultimately, NUE.

Methods

Morphological characteristics, seed yield and metabolite levels of 12 spring barley (Hordeum vulgare) genotypes were compared when grown at high and low nitrogen levels in field conditions during the 2007 and 2008 Canadian growing seasons, and in potted and hydroponic growth-chamber conditions. Genotypic NUpE, NUtE and NUE were calculated and compared between field and growth-chamber environments.

Key Results

Growth chamber and field tests generally showed consistent NUE characteristics. In the field, Vivar, Excel and Ponoka, showed high NUE phenotypes across years and N levels. Vivar also had high NUE in growth-chamber trials, showing NUE across complex to simplistic growth environments. With the high NUE genotypes grown at low N in the field, NUtE predominates over NUpE. N metabolism-associated amino acid levels were different between roots (elevated glutamine) and shoots (elevated glutamate and alanine) of hydroponically grown genotypes. In field trials, metabolite levels were different between Kasota grown at high N (elevated glutamine) and Kasota at low N plus Vivar at either N condition.

Conclusions

Determining which trait(s) or gene(s) to target to improve barley NUE is important and can be facilitated using simplified growth approaches to help determine the NUE phenotype of various genotypes. The genotypes studied showed similar growth and NUE characteristics across field and growth-chamber tests demonstrating that simplified, low-variable growth environments can help pinpoint genetic targets for improving spring barley NUE.     

Evolution after Introduction of a Novel Metabolic Pathway Consistently Leads to Restoration of Wild-Type Physiology

Organisms cope with physiological stressors through acclimatizing mechanisms in the short-term and adaptive mechanisms over evolutionary timescales. During adaptation to an environmental or genetic perturbation, beneficial mutations can generate numerous physiological changes: some will be novel with respect to prior physiological states, while others might either restore acclimatizing responses to a wild-type state, reinforce them further, or leave them unchanged. We examined the interplay of acclimatizing and adaptive responses at the level of global gene expression in Methylobacterium extorquens AM1 engineered with a novel central metabolism. Replacing central metabolism with a distinct, foreign pathway resulted in much slower growth than wild-type. After 600 generations of adaptation, however, eight replicate populations founded from this engineered ancestor had improved up to 2.5-fold. A comparison of global gene expression in wild-type, engineered, and all eight evolved strains revealed that the vast majority of changes during physiological adaptation effectively restored acclimatizing processes to wild-type expression states. On average, 93% of expression perturbations from the engineered strain were restored, with 70% of these occurring in perfect parallel across all eight replicate populations. Novel changes were common but typically restricted to one or a few lineages, and reinforcing changes were quite rare. Despite this, cases in which expression was novel or reinforced in parallel were enriched for loci harboring beneficial mutations. One case of parallel, reinforced changes was the pntAB transhydrogenase that uses NADH to reduce NADP⁺ to NADPH. We show that PntAB activity was highly correlated with the restoration of NAD(H) and NADP(H) pools perturbed in the engineered strain to wild-type levels, and with improved growth. These results suggest that much of the evolved response to genetic perturbation was a consequence rather than a cause of adaptation and that physiology avoided “reinventing the wheel” by restoring acclimatizing processes to the pre-stressed state.     

Simple method for locating a suitable venipuncture site on the tail of the Virginia opossum (Didelphis virginiana)

We identified a site suitable for venipuncture on the tail of the Virginia opossum (Didelphis virginiana) that is reliably and easily located. The prominent hemal arch associated with the ventral surface of caudal vertebra 5 serves as an easily palpated anatomical landmark for locating the ventral caudal vein for blood collection. Because this venipuncture site is only thinly covered by fur and visualization of the vein is not necessary for its location, site preparation and total animal handling time for routine venipuncture are minimal. Blood may be collected from immature and adult male and female animals, and the technique is easily taught to new technicians with minimal danger of injury to the animal.     

Respiratory cycle related EEG changes: Modified respiratory cycle segmentation

Respiratory cycle related EEG change (RCREC) is characterized by significant relative EEG power changes within different stages of respiration during sleep. RCREC has been demonstrated to predict sleepiness in patients with obstructive sleep apnoea and is hypothesized to represent microarousals. As such RCREC may provide a sensitive marker of respiratory arousals. A key step in quantification of RCREC is respiratory signal segmentation which is conventionally based on local maxima and minima of the nasal flow signal. We have investigated an alternative respiratory cycle segmentation method based on inspiratory/expiratory transitions. Sixty two healthy paediatric participants were recruited through staff of local universities in Bolivia. Subjects underwent attended polysomnography on a single night (Compumedics PS2 system). Studies were sleep staged according to standard criteria. C3/A2 EEG channel and time-locked nasal flow (thermistor) were used in RCREC quantification. Forty Seven subjects aged 7–17 (11.4 ± 3) years (24M:23F) were found to have usable polysomnographs for the purpose of RCREC calculation. Respiratory cycles were segmented using both the conventional and novel (transition) methods and differences in RCREC derived from the two methods were compared in each frequency band. Significance of transition RCREC as measured by Fisher's F value through analysis of variance (ANOVA) was found to be significantly higher than the conventional RCREC in all frequency bands (P < 0.05) but beta. This increase in statistical significance of RCREC as demonstrated with the novel transition segmentation approach suggests better alignment of the respiratory cycle segments with the underlying physiology driving RCREC.     

Pre-Treatment with Amifostine Protects against Cyclophosphamide-Induced Disruption of Taste in Mice

Cyclophosphamide (CYP), a commonly prescribed chemotherapy drug, has multiple adverse side effects including alteration of taste. The effects on taste are a cause of concern for patients as changes in taste are often associated with loss of appetite, malnutrition, poor recovery and reduced quality of life. Amifostine is a cytoprotective agent that was previously shown to be effective in preventing chemotherapy-induced mucositis and nephrotoxicity. Here we determined its ability to protect against chemotherapy-induced damage to taste buds using a mouse model of CYP injury. We conducted detection threshold tests to measure changes in sucrose taste sensitivity and found that administration of amifostine 30 mins prior to CYP injection protected against CYP-induced loss in taste sensitivity. Morphological studies showed that pre-treatment with amifostine prevented CYP-induced reduction in the number of fungiform taste papillae and increased the number of taste buds. Immunohistochemical assays for markers of the cell cycle showed that amifostine administration prevented CYP-induced inhibition of cell proliferation and also protected against loss of mature taste cells after CYP exposure. Our results indicate that treatment of cancer patients with amifostine prior to chemotherapy may improve their sensitivity for taste stimuli and protect the taste system from the detrimental effects of chemotherapy.     

Heritable Gene Knockout in Caenorhabditis elegans by Direct Injection of Cas9–sgRNA Ribonucleoproteins

We present a novel method of targeted gene disruption that involves direct injection of recombinant Cas9 protein complexed with guide RNA into the gonad of the nematode Caenorhabditis elegans. Biallelic mutants were recovered among the F₁ progeny, demonstrating the high efficiency of this method.