Agroinfection and nucleotide sequence of cloned wheat dwarf virus DNA

Cloned DNA of the geminivirus wheat dwarf virus (WDV) was successfully used to infect seedling wheat plants. The clone was derived from circular double-stranded viral DNA isolated from naturally infected tissue. The initiation of infection was mediated by Agrobacterium tumefaciens using cloned dimeric WDV genomes in a binary Agrobacterium vector. The WDV DNA which comprised the infectious clone was sequenced and is compared with the published sequence of a Swedish isolate of the same virus. The results confirm that the single WDV genome component of 2.75 kb carries all the information necessary for production of viral symptoms, virus particles and viral double- and single-stranded DNA forms.     

The proton pore in the Escherichia coli F0F1-ATPase: substitution of glutamate by glutamine at position 219 of the alpha-subunit prevents F0-mediated proton permeability

Three mutations in the uncB gene encoding the a-subunit of the F0 portion of the F0F1-ATPase of Escherichia coli were produced by site-directed mutagenesis. These mutations directed the substitution of Glu-219 by Gln, or of Lys-203 by Ile, or of Glu-196 by Ala. Strains carrying either the Lys-203 or Glu-196 substitutions showed growth characteristics indistinguishable from the coupled control strain. Properties of membrane preparations from these strains were also similar to those from the coupled control strain. The substitution of Glu-219 by Gln resulted in a strain which was unable to utilise succinate as sole carbon source and had a growth-yield characteristic of an uncoupled strain. Membrane preparations of the Glu-219 mutant were proton impermeable and the F1-ATPase activity was inhibited by about 50% when membrane-bound. The results are discussed with reference to a previously proposed intramembranous proton pore involving subunits a and c.     

Effects of hyperoxia on composition and rate of synthesis of fatty acids in Escherichia coli

Growth of and fatty acid synthesis in Escherichia coli were inhibited by oxygen at partial pressures above 1 atm and were prevented by exposure to oxygen at 4.2 atm on membranes incubated on a minimal medium. Growth and fatty acid synthesis returned to control rates when cells were removed from hyperoxia to air. The spectrum of fatty acids produced was unchanged by oxygen at pressures which reduced the rate of synthesis. In situ fatty acids were stable to oxygen at pressures which prevented growth and synthesis. Reinitiation of synthesis after complete inhibition in hyperoxia occurred without production of aberrant fatty acids. Fatty acid synthetase specific activity was virtually unchanged, compared with air controls, in cells exposed either to 3.2 or to 15.2 atm of oxygen. The spectrum of fatty acids synthesized by cell-free extracts during incubation in 4.2 atm of oxygen was not different from air-incubated controls. Synthetase assays included added NADPH, acyl carrier protein, mercaptoethanol, and malonyl coenzyme A; hence, damage, other than reversible sulfhydryl oxidation, to the apoenzymes of synthetase was ruled out.     

Beguiling and risky: ‘environmental works and measures’ for wetland conservation under a changing climate

In Australia’s Murray–Darling Basin, small-scale engineering works called ‘environmental works and measures’ have been implemented as a basis for river and other wetland conservation. While implementing these, governments seem to have embraced the beguiling notion that scarce water supplies can be divided further, while conserving the environment and maintaining agricultural production. The difficulties in doing this are expected to increase in the face of extreme climate variability. With this scenario as a backdrop, the $280 million (Monetary values ($) in this paper are in Australian dollars (AUD). At the time of writing AUD $1.00 = ~USD $1.02.) Living Murray and related programmes are assessed to see whether microengineering works to manage the hydrology of wetlands make for effective adaptation to water scarcity and climate change or whether it amounts to an overly narrow adaptation or maladaptation. Some measures were found to be substantially beneficial, such as the construction of fishways. However, under these programmes, only 0.6% of the Basin’s wetlands would be inundated and there are significant risks including desiccation of non-target wetlands and further reductions in water allocations for the environment. It is recommended that trade-offs between alternative strategies are assessed as the basis for minimising perverse impacts under changing climatic and hydrological conditions.     

Measuring Hordein (Gluten) in Beer – A Comparison of ELISA and Mass Spectrometry

Background

Subjects suffering from coeliac disease, gluten allergy/intolerance must adopt a lifelong avoidance of gluten. Beer contains trace levels of hordeins (gluten) which are too high to be safely consumed by most coeliacs. Accurate measurement of trace hordeins by ELISA is problematic.

Methods

We have compared hordein levels in sixty beers, by sandwich ELISA, with the level determined using multiple reaction monitoring mass spectrometry (MRM-MS).

Results

Hordein levels measured by ELISA varied by four orders of magnitude, from zero (for known gluten-free beers) to 47,000 µg/mL (ppm; for a wheat-based beer). Half the commercial gluten-free beers were free of hordein by MS and ELISA. Two gluten-free and two low-gluten beers had zero ELISA readings, but contained significant hordein levels (p<0.05), or near average (60–140%) hordein levels, by MS, respectively. Six beers gave false negatives, with zero ELISA readings but near average hordein content by MS. Approximately 20% of commercial beers had ELISA readings less than 1 ppm, but a near average hordein content by MS. Several barley beers also contained undeclared wheat proteins.

Conclusions

ELISA results did not correlate with the relative content of hordein peptides determined by MS, with all barley based beers containing hordein. We suggest that mass spectrometry is more reliable than ELISA, as ELISA enumerates only the concentration of particular amino-acid epitopes; this may vary between different hordeins and may not be related to the absolute hordein concentration. MS quantification is undertaken using peptides that are specific and unique, enabling the quantification of individual hordein isoforms. This outlines the problem of relying solely on ELISA determination of gluten in beverages such as beer and highlights the need for the development of new sensitive and selective quantitative assay such as MS.     

Engineering high α‐amylase levels in wheat grain lowers Falling Number but improves baking properties

Late maturity α‐amylase (LMA) and preharvest sprouting (PHS) are genetic defects in wheat. They are both characterized by the expression of specific isoforms of α‐amylase in particular genotypes in the grain prior to harvest. The enhanced expression of α‐amylase in both LMA and PHS results in a reduction in Falling Number (FN), a test of gel viscosity, and subsequent downgrading of the grain, along with a reduced price for growers. The FN test is unable to distinguish between LMA and PHS; thus, both defects are treated similarly when grain is traded. However, in PHS‐affected grains, proteases and other degradative process are activated, and this has been shown to have a negative impact on end product quality. No studies have been conducted to determine whether LMA is detrimental to end product quality. This work demonstrated that wheat in which an isoform α‐amylase (TaAmy3) was overexpressed in the endosperm of developing grain to levels of up to 100‐fold higher than the wild‐type resulted in low FN similar to those seen in LMA‐ or PHS‐affected grains. This increase had no detrimental effect on starch structure, flour composition and enhanced baking quality, in small‐scale 10‐g baking tests. In these small‐scale tests, overexpression of TaAmy3 led to increased loaf volume and Maillard‐related browning to levels higher than those in control flours when baking improver was added. These findings raise questions as to the validity of the assumption that (i) LMA is detrimental to end product quality and (ii) a low FN is always indicative of a reduction in quality. This work suggests the need for a better understanding of the impact of elevated expression of specific α‐amylase on end product quality.     

Arabidopsis ammonium transporters,AtAMT1;1 and AtAMT1;2, have different biochemical properties and functional roles

We have compared the biochemical properties of two different Arabidopsis ammonium transporters, AtAMT1;1 and AtAMT1;2, expressed in yeast, with the biophysical properties of ammonium transport in planta. Expression of the AtAMT1;1 gene in Arabidopsis roots increased approximately four-fold in response to nitrogen deprivation. This coincided with a similar increase in high-affinity ammonium uptake by these plants. The biophysical characteristics of this high-affinity system (K_m for ammonium and methylammonium of 8 M and 31 M, respectively) matched those of AtAMT1;1 expressed in yeast (K_m for methylammonium of 32 M and K_i for ammonium of 1–10 M). The same transport system was present, although less active, in nitrate-fed roots. Ammonium-fed plants exhibited the lowest rates of ammonium uptake and appeared to deploy a different transporter (K_m for ammonium of 46 M). Expression of AtAMT1;2 in roots was insensitive to changes in nitrogen nutrition. In contrast to AtAMT1;1, AtAMT1;2 expressed in yeast exhibited biphasic kinetics for methylammonium uptake: in addition to a high-affinity phase with a K_m of 36 M, a low-affinity phase with a K_m for methylammonium of 3.0 mM was measured. Despite the presence of a putative chloroplast transit peptide in AtAMT1;2, the protein was not imported into chloroplasts in vitro. The electrophysiological data for roots, together with the biochemical properties of AtAMT1;1 and Northern blot analysis indicate a pre-eminent role for AtAMT1;1 in ammonium uptake across the plasma membrane of nitrate-fed and nitrogen-deprived root cells.