生物化学

The bacterial flagellar type III export apparatus consists of a cytoplasmic ATPase complex and a transmembrane export gate complex, which are powered by ATP and proton motive force (PMF) across the cytoplasmic membrane, respectively, and transports flagellar component proteins from the cytoplasm to the distal end of the growing flagellar structure where their assembly occurs (Minamino, 2014). The export gate complex can utilize sodium motive force in addition to PMF when the cytoplasmic ATPase complex does not work properly. A transmembrane export gate protein FlhA acts as a dual ion channel to conduct both H⁺ and Na⁺ (Minamino et al., 2016). Here, we describe how to measure the intracellular Na⁺ concentrations in living Escherichia coli cells using a sodium-sensitive fluorescent dye, CoroNa Green (Minamino et al., 2016). Fluorescence intensity measurements of CoroNa Green by epi-fluorescence microscopy allows us to measure the intracellular Na⁺ concentration quantitatively.

Salt stress is a major issue for plants growing in both natural and agricultural settings (Deinlein et al., 2014). For example, irrigation can lead to the build up of salts in the soil as the irrigation water evaporates, leading to salinization, inhibition of plant growth, reduced productivity and eventually to loss of agriculturally usable land. One key element in trying to understand how salt stress impacts plant growth and development, in defining plant salt sensing and response mechanisms and eventually in the breeding or engineering of plants resistant to this stress is monitoring their salt uptake and redistribution. Methods such as imaging Na-sensitive fluorescent probes (Kader and Lindberg, 2005) and use of Na-ion selective microelectrodes (Shabala et al., 2005) offer the potential to follow Na levels in the plant in a non-destructive manner but are technically demanding and not applicable to field, or even many laboratory, conditions. However, tissue sampling followed by inductively coupled plasma spectroscopy (ICP) represents a simple, quantitative assay to monitor total Na levels in plant samples. ICP analysis is also applicable to plants in any environment where samples can be harvested. The approach uses tissue digestion in acid solutions, followed by injection of the resulting sample into an inductively coupled plasma spectrometer and monitoring the characteristic emitted spectrum from Na. As Na is stable, no complex sample preservation is required. Care needs to be taken with possible Na contamination in standards and samples from the water used for sample preparation and from glassware but otherwise, the approach is simple and robust.