Remodelling of arabinoxylan in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) endosperm cell walls during grain filling

Previous studies using spectroscopic imaging have allowed the spatial distribution of structural components in wheat endosperm cell walls to be determined. FT-IR microspectroscopy showed differing changes in arabinoxylan (AX) structure, during grain development under cool/wet and hot/dry growing conditions, for differing cultivars (Toole et al. in Planta 225:1393–1403, 2007). These studies have been extended using Raman microspectroscopy, providing more details of the impact of environment on the polysaccharide and phenolic components of the cell walls. NMR studies provide complementary information on the types and levels of AX branching both early in development and at maturity. Raman microspectroscopy has allowed the arabinose:xylose (A/X) ratio in the cell wall AX to be determined, and the addition of ferulic acid and related phenolic acids to be followed. The changes in the A/X ratio during grain development were affected by the environmental conditions, with the A/X ratio generally being slightly lower for samples grown under cool/wet conditions than for those from hot/dry conditions. The degree of esterification of the endosperm cell walls with ferulic acid was also affected by the environment, being lower under hot/dry conditions. The results support earlier suggestions that AX is either delivered to the cell wall in a highly substituted form and is remodelled through the action of arabinoxylan arabinofuranohydrolases or arabinofuranosidases, or that low level substituted AX are incorporated into the wall late in cell wall development, reducing the average degree of substitution, and that the rate of this remodelling is influenced by the environment. ¹H NMR provided a unique insight into the chemical structure of intact wheat endosperm cell walls, providing qualitative information on the proportions of mono- and disubstituted AX and the levels of branching of adjacent units. The A/X ratio did not change greatly with either the development stage or the growth conditions, but the ratio of mono- to disubstituted Xylp residues increased markedly (by about fourfold) in the more mature samples, confirming the changes in branching levels determined using FT-IR. To the best of our knowledge, this is the first time that intact endosperm cell walls have been studied by ¹H NMR.     

Temporal and spatial changes in cell wall composition in developing grains of wheat cv. Hereward

A combination of enzyme mapping, FT-IR microscopy and NMR spectroscopy was used to study temporal and spatial aspects of endosperm cell wall synthesis and deposition in developing grain of bread wheat cv. Hereward. This confirmed previous reports that changes in the proportions of the two major groups of cell wall polysaccharides occur, with β-glucan accumulating earlier in development than arabinoxylan. Changes in the structure of the arabinoxylan occurred, with decreased proportions of disubstituted xylose residues and increased proportions of monosubstituted xylose residues. These are likely to result, at least in part, from arabinoxylan restructuring catalysed by enzymes such as arabinoxylan arabinofurano hydrolase and lead to changes in cell wall mechanical properties which may be required to withstand stresses during grain maturation and desiccation.     

Factors affecting the robustness of metabolite fingerprinting using 1H NMR spectra

1H NMR spectroscopy is one of the techniques whose potential is currently being explored in the emerging field of metabolomics. It is a non-targeted method, producing signals for all proton-containing chemical species. For crude plant materials the spectra are always complex, with many signals overlapping. Hence a most suitable approach for analysing them is 'metabolite fingerprinting', which is aimed at highlighting compositional similarities and exploring the overall natural variability in a population of samples. The most commonly used method for this is principal component analysis (PCA), as it allows the whole spectral trace to be analysed and the vast quantity of information to be simplified. In this paper we investigate whether there are factors which may affect the NMR spectra in a way that subsequently decreases the robustness of the metabolite fingerprinting by PCA. Imperfections in the signal registration (i.e. inconsistency of the peak position) are generally detrimental to analysing whole traces by multivariate methods. The sources of such problems are illustrated through specially designed repeatability studies using potato and tomato samples, and the analysis of a tea dataset containing many samples. Careful sample preparation can help to limit peak shifts; for instance here by attempting to control the pH of the extracts. In addition, some compounds are susceptible to interactions affecting their chemical shifts and mathematical alignment of peaks may be necessary. Lastly factors such as resolution can also affect analyses and must be carefully adjusted. Our choice of examples aims to raise awareness of potential problems. We do not question the validity of the NMR approach, but point out those areas where special care may need to be taken.     

Stabilization of a pH-sensitive apoptosis-linked coiled coil through single point mutations

The apoptosis-associated Par-4 protein has been implicated in cancers of the prostate, colon, and kidney, and in Alzheimer's and Huntington's diseases, among other neurodegenerative disorders. Previously, we have shown that a peptide from the Par-4 C-terminus, which is responsible for Par-4 self-association as well as interaction with all currently identified effector molecules, is natively unfolded at neutral pH, but forms a tightly associated coiled coil at acidic pH and low temperature. Here, we have alternately mutated the two acidic residues predicted to participate in repulsive electrostatic interactions at the coiled coil interhelical interface. Analysis of circular dichroism spectra reveals that a dramatic alteration of the folding/unfolding equilibrium of this peptide can be effected through directed-point mutagenesis, confirming that the two acidic residues are indeed key to the pH-dependent folding behavior of the Par-4 coiled coil, and further suggesting that alleviation of charge repulsion through exposure to either a low pH microenvironment or an electrostatically complementary environment may be necessary for efficient folding of the Par-4 C-terminus.     

Field trials in Norway with SLICE (0.2% emamectin benzoate) for the oral treatment of sea lice infestation in farmed Atlantic salmon Salmo salar

Four commercial salmon farms on the West coast of Norway were recruited to a programme of field trials in which the efficacy of SLICE (0.2% emamectin benzoate; Schering-Plough Animal Health) was compared with a commercially available product, EKTOBANN (teflubenzuron 2 g kg(-1); Skretting A/S) in treating natural sea lice Lepeophtheirus salmonis infections in Atlantic salmon Salmo salmar L. At each test site, 3 fish pens were treated with each product. In total, nearly 1.2 million first-year-class fish were included in the trial, of which approximately 561,000 received emamectin benzoate at a dosage of 50 microg kg(-1) body wt d(-1), while approximately 610,000 received teflubenzuron at a dosage of 10 mg kg(-1) body wt d(-1). Medicated feed was provided at 0.5% body wt d(-1) over 7 consecutive days. Feed containing emamectin benzoate was generally well accepted by the fish and no problems were encountered in feeding the medicated diet at the desired dose. Lice numbers were counted 2 d before and 1, 7, 14 and 21 d after commencement of treatment. While treatment with both substances rapidly reduced lice numbers, pens treated with emamectin benzoate were found to harbour significantly fewer lice 14 and 21 d post-treatment. Twenty-one days following treatment with emamectin benzoate the lice abundance was reduced on average by 94%. Limited sampling outside the main study period indicated that emamectin benzoate protects against sea-lice infestation over longer periods.     

Development of endothermy in a tasmanian marsupial, Bettongia gaimardi and its response to cold and noradrenaline

Marsupials at birth are ectothermic and gradually attain the ability to change their metabolic heat production during pouch life. How this process occurs in the bettong has been measured on 13 pouch young from week 1 until 3 weeks after pouch vacation (week 18). Oxygen consumption was measured at 35 °C (pouch temperature) and at 22 °C. The results at 35 °C showed an increase in metabolic rate from week 1 until week 12 when there was a decrease to near adult levels after pouch vacation. At 22 °C young bettongs had a lower metabolic rate (compared with measurements made at 35 °C) until week 9 after which there was an increase above measurements made at 35 °C. Noradrenaline had little effect until week 10 after which the metabolic rate (although measured at 35 °C) paralleled the levels measured at 22 °C. The free thyroxine level was low in early pouch life, increased to a peak at week 12 then decreased. Thermal conductance increased until week 10 after which it decreased, reaching values similar to those of adult bettongs by week 20. The results indicate that non-shivering thermogenesis occurs in this macropodoid marsupial. This phenomenon may be a phylogenetic difference between macropodid and non-macropodid marsupials as also suggested by Nicol et al. (1997). Accepted: 19 February 1998