Quantitative X-ray Elemental Imaging in Plant Materials at the Subcellular Level with a Transmission Electron Microscope: Applications and Limitations

Abstract

Energy-dispersive X-ray microanalysis (EDX) is a technique for determining the distribution of elements in various materials. Here, we report a protocol for high-spatial-resolution X-ray elemental imaging and quantification in plant tissues at subcellular levels with a scanning transmission electron microscope (STEM). Calibration standards were established by producing agar blocks loaded with increasing KCl or NaCl concentrations. TEM-EDX images showed that the salts were evenly distributed in the agar matrix, but tended to aggregate at high concentrations. The mean intensities of K+, Cl−, and Na+ derived from elemental images were linearly correlated to the concentrations of these elements in the agar, over the entire concentration range tested (R > 0.916). We applied this method to plant root tissues. X-ray images were acquired at an actual resolution of 50 nm × 50 nm to 100 nm × 100 nm. We found that cell walls exhibited higher elemental concentrations than vacuoles. Plants exposed to salt stress showed dramatic accumulation of Na+ and Cl− in the transport tissues, and reached levels similar to those applied in the external solution (300 mM). The advantage of TEM-EDX mapping was the high-spatial-resolution achieved for imaging elemental distributions in a particular area with simultaneous quantitative analyses of multiple target elements.

NaCl-elicited, vacuolar Ca2+ release facilitates prolonged cytosolic Ca2+ signaling in the salt response of Populus euphratica cells

“…Our previous studies have shown that P. euphratica roots and cell cultures sequestered Na + and Cl − into vacuoles under NaCl stress [30,36,37,51,52]. To determine whether IP 3 /cADPR-stimulated vacuolar Ca 2+ release could mediate the entry of Na + and Cl − into vacuoles, we measured the NaCl-induced H + flux across vacuolar membranes.…”

“…Series 4: effects of salt-elicited vacuolar Ca 2+ release on ionic homeostasis P. euphratica cells were pretreated with neomycin (25 M) and nicotinamide (50 mM) for 2 h, followed by 48 h exposure to NaCl (150 mM) in the absence or presence of inhibitors. Callus cells were collected at four time points (12,24,36, and 48 h) to measure intracellular concentrations of Na + , K + , and Cl − . Moreover, steady-state flux profiles of Na + , K + , Cl − and H + were recorded with NMT.…”

“…Steady-state Na + , H + , K + , and Cl − flux measurements Untreated cells (control) and cells treated with NaCl alone, or with NaCl + antagonists (neomycin, nicotinamide) were sampled at different time points (12,24,36, and 48 h; Series 4). Each cell sample was placed in the center of a poly-l-lysine-pretreated coverslip (2 cm × 2 cm), which was placed in the measuring chamber.…”

“…Populus euphratica is a salt-resistant model species often used to elucidate physiological and molecular mechanisms underlying salt tolerance in woody plants [32][33][34][35][36]. In this study, P. euphratica cell cultures were used as a model system for investigating salt signaling network.…”

Analysis of the Epicuticular Wax Composition of Jasminum Employing Sem-Edx

“…Energy Dispersive X-ray Analysis (EDX) is a technique for analyzing elements at the microscopic level. For this purpose, Scanning (SEM) or Transmission (TEM) electron microscopes are equipped with an energy dispersive system for quantitative electron probe X-ray microanalysis (Chen et al, 2014). For the present study, small pieces of fresh leaf were taken, washed with distilled water, air dried and coated with gold.…”