Plants employ a number of phosphorylation cascades in response to a wide range of environmental stimuli. Previous studies in Arabidopsis and yeast indicate that histidine kinase AHK1 is a positive regulator of drought and osmotic stress responses. Based on these studies AHK1 was proposed a plant osmosensor, although the molecular basis of plant osmosensing still remains unknown. To understand the molecular role and signaling mechanism of AHK1 in osmotic stress, we have expressed and purified full-length AHK1 from Arabidopsis in a bacterial host to allow for studies on the isolated transmembrane receptor. Purification of the recombinant protein solubilized from the host membranes was achieved in a single step by metal-affinity chromatography. Analysis of the purified AHK1 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting show a single band indicating that the preparation is highly pure and devoid of contaminants or degradation products. In addition, gel filtration experiments indicate that the preparation is homogenous and monodisperse. Finally, CD-spectroscopy, phosphorylation activity, dimerization studies, and protein–protein interaction with plant phosphorylation targeting AHP2 demonstrate that the purified protein is functionally folded and acts as phospho-His or phospho-Asp phosphatase. Hence, the expression and purification of recombinant AHK1 reported here provide a basis for further detailed functional and structural studies of the receptor, which might help to understand plant osmosensing and osmosignaling on the molecular level.
Circularity of cellulose-based pre- and post-consumer wastes requires an integrated approach which has to consider the characteristics of the fibre polymer and the presence of dyes and additives from textile chemical processing as well. Fibre-to-fibre recycling is a condition to avoid downcycling of recycled material. For cellulose fibres regeneration via production of regenerated cellulose fibres is the most promising approach. Textile wastes contain dyes and additives, thus a recycling technique has to be robust enough to process such material. In an ideal case the reuse of colorants can be achieved as well. At present nearly 80% of the regenerated cellulose fibre production utilises the viscose process, therefore this technique was chosen to investigate the recycling of dyed material including the reuse of the colorant. In this work, for the first time, a compilation of all required process steps to a complete circular concept is presented and discussed as a model. Indigo-dyed viscose fibres were used as a model to study cellulose recycling via production of regenerated cellulose fibres to avoid downcycling. Indigo was found compatible to the alkalisation and xanthogenation steps in the viscose process and blue coloured cellulose regenerates were recovered from indigo-dyed cellulose. A supplemental addition of reduced indigo to the cellulose solution was also found feasible to adjust colour depth in the regenerated cellulose to the level required for use as warp material in denim production. By combination of fibre recycling and indigo dyeing the conventional yarn dyeing in denim production can be omitted. Model calculations for the savings in water and chemical consumption demonstrate the potential of the process. The proportion of the substitution will depend on the collection rate of denim wastes and on the efficiency of the fibre regeneration process. Estimates indicate that a substitution of more than 70% of the cotton fibres by regenerated cellulose fibres could be achieved when 80% of the pre- and post-consumer denim wastes are collected. Therefore, the introduction of fibre recycling via regenerated cellulose fibres will also make a substantial impact on the cotton consumption for jeans production.
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