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Green, K.

Publications and source records attributed to Green, K..

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Alleviation of Al toxicity by Si is associated with the formation of Al-Si complexes in root tissues of sorghum

Silicon is reported to reduce the toxic effects of Al on root elongation but the in planta mechanism by which this occurs remains unclear. Using seedlings of soybean (Glycine max) and sorghum (Sorghum bicolor), we examined the effect of up to 2 mM Si on root elongation rate (RER) in Al-toxic nutrient solutions. Synchrotron-based low energy X-ray fluorescence (LEXRF) was then used for the in situ examination of the distribution of Al and Si within cross-sections cut from the apical tissues of sorghum roots. The addition of Si potentially increased RER in Al-toxic solutions, with RER being up to ca. 0.3 mm h-1 (14 %) higher for soybean and ca. 0.2 mm h-1 (17 %) higher for sorghum relative to solutions without added Si. This improvement in RER could not be attributed to a change in Al-chemistry of the bulk nutrient solution, nor was it due to a change in the concentration of Al within the apical (0-10 mm) root tissues. Using LEXRF to examine sorghum, it was demonstrated that in roots exposed to both Al and Si, much of the Al was co-located with Si in the mucigel and outer apoplast. These observations suggest that Si reduces the toxicity of Al in planta through formation of Al-Si complexes in mucigel and outer cellular tissues, thereby decreasing the binding of Al to the cell wall where it is known to inhibit wall loosening as required for cell elongation.

plant biology

An improved method for the expression screening of membrane protein-GFP fusions in yeast

The expression and purification of membrane proteins is an extremely challenging area of work within Protein Science. Membrane proteins are required for compound screening and structure determination in industry. Here we describe some new and innovative methodology in developing the membrane protein GFP fusion primary expression screening in yeast. This methodology enables the expression of membrane proteins fused to GFP in both Saccharomyces cerevisiae and Pichia pastoris systems. This capability helps facilitate screening of constructs to establish which are suitable for membrane protein production for compound screening and structure determination\n\nIn terms of the primary screening work, we have developed both agar plate and liquid plate expression methodology in yeast. The two approaches correspond well, but the agar plate method is more rapid and we have shown it to have the advantage of allowing cells to be taken directly into confocal microscopy for immediate cell localisation data. Innovative work to extend the methanol induction time in the Pichia agar plate method established good differentiation from the background. A novel agar plate method was also developed for S.cerevisiae which is also presented. These screening methods allow triaging of constructs for either membrane protein preps for biochemical assays or progression to fluorescence size exclusion chromatography; where various detergents can be screened to determine the most appropriate for membrane protein solubilisation, the starting point for purification, crystallisation and structure determination.\n\nMembrane targets depicted to demonstrate the improved primary screening methodology are a copper transporter Ctr1p from S.cerevisiae and a water transporter Aqp4 from human origin.\n\nHighlightsAn improved method for the production of recombinant MP-GFP fusions in yeast is presented using agar plates.\n\nAn agar plate method for MP-GFP expression screening is described for Pichia pastoris, with improved induction methodology by the simple addition of methanol, allowing longer induction times for expression clarity.\n\nA new simple rapid agar plate method for MP-GFP expression screening is described for Saccharomyces cerevisiae.\n\nCells can be taken directly from agar plates into confocal microscopy studies for immediate cell localisation data and triaging.\n\nLiquid plate based screening methods are also described for both yeasts in comparison, to show there is corresponding data, helping validate the new agar plate methods.

biochemistry