Sodium and calcium localization in cells and tissues by precipitation with antimonate: A quantitative study

Summary Komnick's antimonate technique, which was devised to localize Na⁺ in cells and tissues, was studied quantitatively. Some modifications, as well as its application to Ca²⁺ localization, were also investigated.We combined measurements of Na⁺ and Ca²⁺ retention in plant roots during the various procedures, electron microscopy, autoradiography, and semiquantitative X-ray microanalysis. We were able to show that (at least in barley roots) antimonate does not precipitate at all with Na⁺, irrespective of the Na⁺ content of the tissue or the method of antimonate application. (Even during precipitative freeze dissolution or after freeze drying, no Na⁺ is precipitated.) By means of Komnick's antimonate technique Ca²⁺ is trapped within the tissue, but only after serious dislocation. Perspectives for reliable localization of diffusible ions in cells and tissues, by precipitation simultaneously with conventional fixations, are bad.     

Do vascular plants and bryophytes respond differently to coniferous invasion of coastal heathlands?

Invasion by non-native conifers may pose a threat to local biodiversity, but knowledge about introduced conifer effects on Northern Hemisphere ecosystems is scarce. The coastal heathlands of north-west Europe are threatened by invasion of native and introduced tree species. We assess how spread of the introduced conifer Sitka spruce (Picea sitchensis (Bong.) Carr.) into European coastal heathlands affect two major functional groups; vascular plants and bryophytes, and how these effects relate to the environmental changes imposed by the developing tree canopies. We compared the impact of introduced Sitka spruce and native Scots pine (Pinus sylvestris L.) by analysing effects on species richness and turnover of vascular plants and bryophytes along fine-scale transects from individual tree stems into open heathland vegetation. Environmental impacts were assessed by measured environmental variables, and the responses of the two species groups were assessed by calculating changes in their respective mean Ellenberg indicator values. Species richness decreased beneath both conifers, related to decreased light and increased nitrogen and pH. Whereas vascular plants responded negatively to poor light conditions beneath dense and low Sitka spruce canopies, bryophytes were more negatively affected by the warmer and drier microclimates beneath Scots pine. Introduced Sitka spruce impacts the sub-canopy environment differently from the native Scots pine, and the two functional plant groups responded differently to these impacts. This suggests that future forests are likely to differ in species richness and composition, depending on whether succession is based on native or introduced coniferous trees.     

Catalytic domain of the diversified Pseudomonas syringae type III effector HopZ1 determines the allelic specificity in plant hosts

The type III secretion systems (T3SS) and secreted effectors (T3SEs) are essential virulence factors in Gram‐negative bacteria. During the arms race, plants have evolved resistance (R) genes to detect specific T3SEs and activate defence responses. However, this immunity can be efficiently defeated by the pathogens through effector evolution. HopZ1 of the plant pathogen Pseudomonas syringae is a member of the widely distributed YopJ T3SE family. Three alleles are known to be present in P. syringae, with HopZ1a most resembling the ancestral allelic form. In this study, molecular mechanisms underlying the sequence diversification‐enabled HopZ1 allelic specificity is investigated. Using domain shuffling experiments, we present evidence showing that a central domain upstream of the conserved catalytic cysteine residue determines HopZ1 recognition specificity. Random and targeted mutagenesis identified three amino acids involved in HopZ1 allelic specificity. Particularly, the exchange of cysteine141 in HopZ1a with lysine137 at the corresponding position in HopZ1b abolished HopZ1a recognition in soybean. This position is under strong positive selection, suggesting that the cysteine/lysine mutation might be a key step driving the evolution of HopZ1. Our data support a model in which sequence diversification imposed by the plant R gene‐associated immunity has driven HopZ1 evolution by allowing allele‐specific substrate‐binding.     

Glycine betaine application modifies biochemical attributes of osmotic adjustment in drought stressed wheat

Nineteen wheat genotypes were used to examine the effects of foliar applied glycine betaine (GB, 100 mM) on concentration of various osmolytes (such as proline, choline, GB and sucrose) under drought stress conditions. Drought stress caused a significant increase in proline content and GB content of wheat genotypes, both at maximum tillering and anthesis stages. Choline and sucrose were accumulated significantly at higher levels under stress conditions at both the stages. GB application increased the proline content and endogenous levels of GB in comparison to their stressed counterparts both at maximum tillering and anthesis stages but this increase was observed to be genotype specific. Furthermore, significant decrease in choline levels and sucrose contents of GB treated plants at anthesis stage and enhanced levels of proline questioned about involvement of GB in production of other osmolytes as well as stage specific response of wheat genotypes to GB spray. But these changes in osmolyte accumulation (OA) were not correlated with relative water content and stress tolerance index observed, under both GB sprayed and non-sprayed drought stressed conditions. So OA could not be considered as a selection criteria for drought tolerance in wheat.     

Pseudomonas aeruginosa outer membrane protein OprH: expression from the cloned gene and function in EDTA and gentamicin resistance.

Overexpression of major outer membrane protein OprH of Pseudomonas aeruginosa as a result of mutation (in strain H181) or adaptation to low Mg2+ concentrations (in parent strain H103) is accompanied by increased resistance to polymyxin B, gentamicin, and EDTA. A 2.8-kb EcoRI fragment containing the oprH gene was subcloned into several different expression plasmids in Escherichia coli. These experiments showed that significant levels of OprH could be produced from a promoter on the EcoRI fragment; that the cloned oprH gene was not regulated by Mg2+ deficiency; that there were no differences in the expression of OprH in any construction, regardless of whether the gene from strain H103 or its OprH-overexpressing, polymyxin B-resistant derivative, strain H181, was used; and that overexpression of OprH in E. coli to the level observed in P. aeruginosa H181 did not result in a resistance phenotype. These results favored the conclusion that the mutation in strain H181 was a regulatory rather than a promoter mutation. The oprH gene was cloned behind the benzoate-inducible pm promoter in plasmid pGB25 and transferred to P. aeruginosa H103. Overexpression of OprH from the cloned gene in H103/pGB25 resulted in EDTA resistance but not polymyxin B resistance. This result suggested that another factor, possibly lipopolysaccharide, was affected by the mutation in strain H181. Consistent with this suggestion was the demonstration that mutants of strain H181 with alterations in lipopolysaccharide had reverted to wild-type polymyxin B susceptibility but had unaltered gentamicin and EDTA resistance. These data were consistent with the hypothesis that OprH replaces outer membrane-stabilizing divalent cations.     

NMR backbone resonance assignments of the prodomain variants of BDNF in the urea denatured state

Brain derived neurotrophic factor (BDNF) is a member of the neurotrophin family of proteins which plays a central role in neuronal survival, growth, plasticity and memory. A single Val66Met variant has been identified in the prodomain of human BDNF that is associated with anxiety, depression and memory disorders. The structural differences within the full-length prodomain Val66 and Met66 isoforms could shed light on the mechanism of action of the Met66 and its impact on the development of neuropsychiatric-associated disorders. In the present study, we report the backbone ¹H, ¹³C, and ¹⁵N NMR assignments of both full-length Val66 and Met66 prodomains in the presence of 2 M urea. These conditions were utilized to suppress residual structure and aid subsequent native state structural investigations aimed at mapping and identifying variant-dependent conformational differences under native-state conditions.     

In suspension cultures of Catharanthus roseus the cyanide-resistant pathway is engaged in respiration by excess sugar in combination with phosphate or nitrogen starvation

Suspension cultured cells of Catharanthus roseus were used to study the effects of excess sugar, and phosphate or nitrogen starvation on growth and respiration. Excess sugar resulted in a higher dry weight but the cell number was not significantly higher than in the controls. After inoculation in phosphate-free medium almost no growth was observed, whereas in medium without nitrogen some growth still occurred, indicating that the medium contains more than enough nitrogen and only a limited amount of phosphate. The maintenance respiration in the nitrogen-free medium was lower than in the control, probably as a result of a lower protein production. In phosphate-free medium, however, the maintenance respiration was higher, because the cyanide-resistant pathway was engaged and the P/O ratio was decreased. In Catharanthus roseus cells the cyanide-resistant pathway is engaged upon phosphate or nitrogen starvation in combination with excess sugar, while excess sugar, as such, does not engage the cyanide-resistant pathway. In the presence of excess sugar the rate of glycolysis is adenylate-controlled. It is only in combination with mineral starvation that the rate of glycolysis does not match the capacity of the cytochrome pathway, resulting in an overflow via the alternative pathway. After combination of mineral starvation with a low sugar supply the rate of glycolysis is controlled by both the substrate supply and the adenylates, and the cyanide-resistant pathway is not engaged.