Electrical impedance spectroscopy of plant cells in aqueous buffer media over a wide frequency range of 4 Hz to 20 GHz.

Electrical impedance spectroscopy was performed on suspensions of plant cells in aqueous buffer media over a wide frequency range of 4 Hz to 20 GHz. Custom probes were designed, manufactured, and used for these investigations. Experiments were performed with a custom-made parallel plate probe and impedance analysers in the low-frequency range (4 Hz to 5 MHz), with a custom-made coaxial airline probe and a vector network analyser in the mid-frequency range (100 kHz to 3 GHz), and with a commercial open-ended probe and a vector network analyser in the high-frequency range (200 MHz to 20 GHz). The impedance data acquired were processed in order to eliminate the effects of parasitics and compensate for geometrical differences between the three probes. Following this, the data were fitted to a unified model consisting of the Randles and Debye models. The data were also normalized to a reference measurement, in order to accentuate the effects of cell concentration on the impedance of the suspensions.•The methodology allows for impedance spectroscopy of cell suspensions over a wide frequency range spanning 10 orders of magnitude.•It allows for compensation of parasitics and of geometrical variations between probes, using mathematical techniques.

SlRLK-like is a malectin-like domain protein affecting localization and abundance of LeEIX2 receptor resulting in suppression of EIX-induced immune responses.

The first line of plant defense occurs when a plant pattern recognition receptor (PRR) recognizes microbe-associated molecular patterns. Plant PRRs are either receptor-like kinases (RLKs), which have an extracellular domain for ligand binding, a single-pass transmembrane domain, and an intracellular kinase domain for activating downstream signaling, or receptor-like proteins (RLPs), which share the same overall structure but lack an intracellular kinase domain. The tomato (Solanum lycopersicum) LeEIX2 is an RLP that binds ethylene-inducing xylanase (EIX), a fungal elicitor. To identify LeEIX2 receptor interactors, we conducted a yeast two-hybrid screen and found a tomato protein that we termed SlRLK-like. The interaction of LeEIX2 with SlRLK-like was verified using co-immunoprecipitation and bimolecular fluorescence complementation assays. The defense responses induced by EIX were markedly reduced when SlRLK-like was overexpressed in Nicotiana benthamiana or Nicotiana tabacum, and knockout of SlRLK-like using the CRISPR/Cas9 system increased EIX-induced ethylene production and 1-aminocyclopropane-1-carboxylate synthase (SlACS2) gene expression in tomato. Co-expression of SlRLK-like with LeEIX2 led to a reduction in its abundance, apparently through an endoplasmic reticulum-associated degradation process. Notably, truncation of SlRLK-like protein revealed that the malectin-like domain is sufficient and essential for its function. Moreover, SlRLK-like associated with the RLK FLS2, resulting in its degradation and concomitantly a reduction of the flagellin 22 (flg22)-induced burst of reactive oxygen species. In addition, SlRLK-like co-expression with other RLPs, Ve1 and AtRLP23, also led to a reduction in their abundance. Our findings suggest that SlRLK-like leads to a decreased stability of various PRRs, leading to a reduction in their abundance and resulting in attenuation of defense responses.

Electrical Impedance Spectroscopy of plant cells in aqueous biological buffer solutions and their modelling using a unified electrical equivalent circuit over a wide frequency range: 4Hz to 20 GHz.

A simple, ultra-wide frequency range, equivalent circuit for plant cell suspensions is presented. The model incorporates both the interfacial interactions of the suspension with the electrode, dominant at low frequencies, and the molecule and cell polarization mechanisms dominant at higher frequencies. Such model is useful for plant cell characterization allowing a single set of parameters over >9 orders of magnitude, whilst allows electronic simulations over the whole frequency range using a single model, simplifying the design of electronic systems of integrated plant cell sensors. The model has been experimentally validated in the frequency range of 4 Hz-20 GHz with each component in the circuit representing a physical phenomenon. Various cell concentrations (MSK8 tomato cells in Murashige and Skoog media) have been investigated, showing clear correlations of the cell capacitance increasing within the range of 200-600 pF, whilst cell resistance (R) decreasing within the range of approximately 0.8-3 kΩ within the cell concentration X-Y cells/mL range. This is the first model ever reported that covers such a wide frequency range and includes both interfacial and polarization effects in this simple form.

Integrated electrochemical Chip-on-Plant functional sensor for monitoring gene expression under stress.

The ability to interact with plants, both to sense and to actuate, would open new opportunities for precision agriculture. These interactions can be achieved by using the plant as part of the sensing system. The present work demonstrates real-time monitoring of ß-glucuronidase (GUS) expression in transgenic tobacco plants using its activity as a biomarker for functional sensing. As "proof of concept", we demonstrated GUS enzyme biosensing under constitutive expression in Msk8 tomato cells and transgenic tobacco plants and in heat shock inducible BY2 tobacco cells and tobacco plants. The sensing was done using a three-electrode microchip in Msk8 or BY2 cell culture or in tobacco plant leaves. The electrode microchip was used to transduce the expression of the GUS enzyme by chronoamperometry to a measurable electrical current signal. For the constitutive expression of GUS in Msk8 cells, the system sensitivity was 0.076 mA/mM-cm2 and the limit of detection was 0.1 mM. For the heat shock inducible BY2 cells the GUS enzyme activity was detected 12-26 h after the heat shock was applied (40 °C for 2 h) using two different substrates: p-nitrophenyl-ß-glucuronide (with sensitivity of 0.051 mA/mM-cm2) and phenolphthalein-ß-glucuronide (with sensitivity of 0.029 mA/mM-cm2).