Walker, K. L. | Search bioRxiv

bioRxiv2018

Real-time whole-plant dynamics of heavy metal transport in Arabidopsis halleri and Arabidopsis thaliana by gamma-ray imaging

Heavy metals such as zinc are essential for plant growth, but toxic at high concentrations. Despite our knowledge of the molecular mechanisms of heavy metal uptake by plants, experimentally addressing the real-time whole-plant dynamics of heavy metal uptake and partitioning has remained a challenge. To overcome this, we applied a high sensitivity gamma-ray imaging system to image uptake and transport of radioactive 65Zn in whole-plant assays of Arabidopsis thaliana and the Zn hyperaccumulator A. halleri. We show that our system can be used to quantitatively image and measure uptake and root-to-shoot translocation dynamics of zinc in real time. In the metal hyperaccumulator Arabidopsis halleri, 65Zn uptake and transport from its growth media to the shoot occurs rapidly and on time scales similar to those reported in rice. In transgenic A. halleri plants in which expression of the zinc transporter gene HMA4 is suppressed by RNAi, 65Zn uptake is completely abolished.\n\nHIGHLIGHTWe have used gamma-ray imaging to visualize the stark differences of real-time whole-plant dynamics of zinc root-to-shoot transport in heavy metal hyperaccumulating and non-accumulating Arabidopsis.

plant biology↗

bioRxiv2018

Optimisation of a novel method for the production of single-span membrane proteins in Escherichia coli

The large-scale production and isolation of recombinant protein is a central element of the biotechnology industry and many of the products have proved extremely beneficial for therapeutic medicine. Escherichia coli is the microorganism of choice for the expression of heterologous proteins for therapeutic application, and a range of high-value proteins have been targeted to the periplasm using the well characterised Sec protein export pathway. More recently, the ability of the second mainstream protein export system, the twin-arginine translocase, to transport fully-folded proteins into the periplasm of not only E. coli, but other Gram-negative bacteria, has captured the interest of the biotechnology industry.\n\nIn this study, we have used a novel approach to block the export of a heterologous Tat substrate in the later stages of the export process, and thereby generate a single-span membrane protein with the soluble domain positioned on the periplasmic side of the inner membrane. Biochemical and immuno-electron microscopy approaches were used to investigate the export of human growth hormone by the twin-arginine translocase, and the generation of a single span membrane-embedded variant. This is the first time that a bona-fide biotechnologically-relevant protein has been exported by this machinery and visualised directly in this manner. The data presented here demonstrate a novel method for the production of single-span membrane proteins in E. coli.\n\nHighlightsO_LIThe Tat translocase has captured the interest of the biotechnology industry\nC_LIO_LIBiochemical and immuno-EM approaches showed efficient export of hGH by Tat\nC_LIO_LIA novel approach was used to block export of hGH by Tat in E. coli\nC_LIO_LIWe demonstrate a novel method for producing single-span membrane proteins in E. coli\nC_LI

biochemistry↗