High Spatial Recording And Analyzing Of Electrical Signal In Plant

Considering as the major study point of plant electrophysiology, electrical signal in plant is the initial response to environmental stimuli. It play an important role in inter-and extracellular communication, and the regulation of physiological processes in higher plants. By now, many considerable progresses have been made in the understanding of electrical signal in plant, but some domains of electrical signal in plant are still lack of studying, especially in the cellular network level. This is in large part due to the technical difficulties of recording electrical activity from large numbers of cells simultaneously, and the few applications of signal processing methods in analyzing the recoded signals. To obtain new comprehensions of plant electrophysiological processes at the cellular network, based on the traditional methods such as extracellular and intracellular recoding, the new techniques multi-electrode array (MEA) and optical recording methods, which can record the electrical activities of multi cells simultaneously, are used to study the electrical signals in higher plants in this research, and some signal processing methods as independent component analysis (ICAX cluster analysis, coherence analysis and information entropy are also used. The main results and conclusions are as follows:1. Combined the recording results and independent component analysis, coherence analysis methods, the source signals of action potential were determined, which proved the mixture character of electrical signal in plant. Using Ag/AgCl electrode and glass microelectrode, the action potentials (APs) in the stem of cucumber induced by electrical stimuli with different intensities were recorded. The recording results showed the amplitude and duration time of action potentials are not same under different intensity stimuli. By plotting the strength-duration curves for the evoked APs, three distinguishable rheobases and chronaxies were found, which suggest there are different threshold strengths for cells in the cucumber stem. The simultaneously intracellular and extracellular recording verified the quantitative relation between intra and extra signal, meanwhile indicated the mixture character of extracellular recording signal. The ICA analysis to APs showed there are different source signals, which can be distinguished by the duration times among them. The cluster analysis to the independent components (ICs) from8samples and128ICs from32samples indicated there are3main clusters for the source signals, which means the APs are composed by3different sources. Considering the recording results, the sieve tube element, companion cell and parenchyma cell are considered as the three types cells that participating in APs.2. Using the MEA technique, we recorded the potential change in vivo in situ at multi sites simultaneously. And found the complex transmitting route of variation potential in the leaf of H. annuus.For the using of multi-electrode array technique, one MEA system MED64was introduced. And methods for the preparation of array, the coupling of H. annuus leaves with MED64planar microelectrodes were given. By comparing the signal/noise ratio under different conducting medium including1mM,2mM,5mM,10mM KC1and buffer solution, the proper medium in the recording chamber was determined. In the experiments, the electrical activities induced by heat stimuli, cooling stimuli and cut stimuli were successfully recorded in64different points of H. annuus leaf using MED64system. To visualize the electrical signal transmitting process, an algorithm was designed. By colour-coding for the electrical onset and mapping the voltage distributions, different transmitting routes including main route and branch route were found for variation potential (VP) in the leaf. The signal transmitting velocity is about lmm/s in the main route and2-3mm/s in the branch route. After a view to the picture obtained with the microscope, a conclusion was determined that the main route mainly follows the main vein in the leaf, and the branch route extends in the region which is composed of epidermal cells, guard cells and mesophyll cells. After the inhibition of ion channels by La3+, TEA, and sodium orthovanadate, which are the blockers of Ca2+channel, K+channel and H+-ATPase, respectively, the amplitude and transmitting of variation potential were reduced. That indicates the potential transmuting is associated with the ion channel and may have an electrical coupling transmitting mode.3. By combining a fluorescence microscope and cold CCD cameras, we built an optical recording system, which can record the fluorescence signal from numbers of cells in the microscope view. To reflect the membrane voltage change, the voltage-sensitive dye DiBAC4(3) was used. To obtain the ΔF/F change from series of fluorescence images, the algorithm was designed and a calculating tool was programmed based on Matlab software. Dye calibration with protoplasts showed the dye fluorescence ΔF/F is about1%when the membrane depolarized lmV in the range of-100mV to0mV, and dye calibration in vivo in the H. annuus stem showed AF/F is about1%when the membrane depolarized10mV in the range of-120mV to0mV. The influence of solution pH change to dye fluorescence was determined. After the calibration, the action potentials induced by electrical stimuli were recorded, meanwhile a high-resolution visualizing for the transmitting of APs were done. The results indicted the APs in the stem of H. annuus mainly distributed in the phloem region, and there are no obvious APs in the xylem, pith or cortex region. The coherence analysis suggested that the APs in the phloem of the H. annuus stem may be synchronized in the observed sites and that there may be a complex information transfer mode for higher plants. And this result provides strong evidence that there are low-resistance bridges that allow AP transmission from cell to cell. For the optical recording in the leaf of H. annuus, we found VPs can be induced in the epidermal cell, guard cell and mesophyll cell, and the information entropy indicated there is information flow in the guard cell region, and also between the guard cell and epidermal cell.