Key Technique Of Non-invasive Ion Flux Recording And Analyzing In Crop Under Abiotic Stress

Plant electrical signals are probably one of the initial responses to external stimulus. It has been reported in China and abroad that plasma membrane potential changed with abiotic stress in plants. Plasma membrane potential change is associated with the ion fluxes of transmembrane. However in fact, the ion mechanism of change in plasma membrane potential is still ambiguous based on the present knowledge. Finding an effective detection technique will have a positive significance. In this study ion mechanism with electrical signals changing in plant was explored combining the electrophysiological intracellular measurement technique with non-invasive extracellular ion flux dynamic detection technique. And taking four varieties of wheat in salt tolerance as examples of higher crops, plasma membrane potentials and dynamic influxes and effluxes of K+ and H+ from wheat root under salt stress were synchronous recorded. Nine feature parameters responding to salt stress were extracted from membrane potentials and ion fluxes, and used to establish a wheat salt-tolerant feature identification model. It provides scientific evidence for using electrical signal to early evaluate the tolerance of abiotic stress. The main work is as follows:(1) Microelectrodes used in membrane potential and extracellular ion flux recording technique were fabricated and performance tested detaily. Three kinds of ion selective microelectrode used in detection of extracellular single ion flux, intracellular ion activity, and extracellular double ions fluxes were fabricated. This fabrication included drawing from glass micropipette, silanization with dimethyldichlorosilane, and filling with internal filling solution and liquid ion exchangers. The performance of ion selective microelectrode such as resistance, Nernstian slope, Nernstian response range, etc.(2) Synchronous recording system of membrane potential and extracellular ion flux was establised. A calibration model and key technology were studied to verify the accuracy and reliability of extracellular ion flux system. Results showed that the noise level of membrane potential and ion flux were within 0.1 mV and 15 μV, separately, which could meet precision request of plant electrical signals recording. A linear interpolation method and a noise reduction algorithm were designed to apply for membrane potential and ion flux data. Experiment demonstrated three ways of implementation of the salt shock of wheat samples, and best one was used.(3) Change synchronously of membrane potential and extracellular ion fluxes of wheat root under salt stress were studied. To evauate salt tolerance of wheat, four varieties of wheat were used in this study, including Dekang 961, Xuezao, Nongda5182 and Jing411. For each wheat, membrane potential and extracellular K+ or H+ fluxes of wheat root under salt stress were recording synchronously. Results showed that there was a positive correlation between hyperpolarization of membrane potential and extracellular H+ fluxes, and this positive correlation was showed in repolarization of membrane potential and extracellular K+ fluxes, too. This study means that probably proton pump involves in the process of hyperpolarization in membrane potential, and repolarization after hyperpolarization is mainly composed of K+ loss contribution. This recording method can help to reveals ion mechanism of salt tolerance in wheat.(4) Kinetic curve feature parameters of salt tolerance in wheat were extracted and a wheat salt-tolerant feature identification model was established. Membrane potential curve and extracellular K+ or H+ fluxes curve under salt stress in four different wheat were plotted.9 kinetic curve feature parameters of salt tolerance in wheat responding to salt stress were extracted to represent the response mechanism of wheat root membrane potential curve and extracellular K+ or H+ fluxes to salt shock as 100 mmol/L NaCl. With kinetic curve feature parameters based on the response to salt stress, a wheat salt-tolerant feature identification model was established base on support vector machine, which can provide methods for research of crop tolerance.