SignificanceNumerous modes of long-distance electrical signaling exist in nature. The best known of these, axonal conduction, requires one primary cell population, i.e., neurons. In contrast, the cell types that mediate leaf-to-leaf electrical signaling in wounded plants have not been defined rigorously. Using genetic approaches, we find that two distinct populations of cells in the vasculature matrix are needed to perform this function. Surprisingly, these cells do not contact each other directly. As we further defined the plant wound response, we found that wound-induced membrane depolarizations preceded large intravasculature calcium fluxes. We reveal a two-cell-type mode of electrical signaling in leaves and discuss parallels and differences in electrical signaling outside the plant kingdom.

Plants are exposed to cellular damage by mechanical stresses, herbivore feeding, or invading microbes. Primary wound responses are communicated to neighboring and distal tissues by mobile signals. In leaves, crushing of large cell populations activates a long-distance signal, causing jasmonate production in distal organs. This is mediated by a cation channel-mediated depolarization wave and is associated with cytosolic Ca 2+ transient currents. Here, we report that much more restricted, single-cell wounding in roots by laser ablation elicits non-systemic, regional surface potential changes, calcium waves, and reactive oxygen species (ROS) production. Surprisingly, laser ablation does not induce a robust jasmonate response, but regionally activates ethylene production and ethylene-response markers. This ethylene activation depends on calcium channel activities distinct from those in leaves, as well as a specific set of NADPH oxidases. Intriguingly, nematode attack elicits very similar responses, including membrane depolarization and regional upregulation of ethylene markers. Moreover, ethylene signaling antagonizes nematode feeding, delaying initial syncytial-phase establishment. Regional signals caused by single-cell wounding thus appear to constitute a relevant root immune response against small invaders.

Jasmonates are oxygenated lipids (oxylipins) that control defense gene expression in response to cell damage in plants. How mobile are these potent mediators within tissues? Exploiting a series of 13-lipoxygenase (13-lox) mutants in Arabidopsis (Arabidopsis thaliana) that displays impaired jasmonic acid (JA) synthesis in specific cell types and using JA-inducible reporters, we mapped the extent of the transport of endogenous jasmonates across the plant vegetative growth phase. In seedlings, we found that jasmonate (or JA precursors) could translocate axially from wounded shoots to unwounded roots in a LOX2-dependent manner. Grafting experiments with the wild type and JA-deficient mutants confirmed shoot-to-root oxylipin transport. Next, we used rosettes to investigate radial cell-to-cell transport of jasmonates. After finding that the LOX6 protein localized to xylem contact cells was not wound inducible, we used the lox234 triple mutant to genetically isolate LOX6 as the only JA precursor-producing LOX in the plant. When a leaf of this mutant was wounded, the JA reporter gene was expressed in distal leaves. Leaf sectioning showed that JA reporter expression extended from contact cells throughout the vascular bundle and into extravascular cells, revealing a radial movement of jasmonates. Our results add a crucial element to a growing picture of how the distal wound response is regulated in rosettes, showing that both axial (shoot-to-root) and radial (cell-to-cell) transport of oxylipins plays a major role in the wound response. The strategies developed herein provide unique tools with which to identify intercellular jasmonate transport routes.

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Since the mechanical properties of single cells together with the intercellular adhesive properties determine the macro-mechanical properties of plants, a method for evaluation of the cell elastic properties is needed to help explanation of the behavior of fruits and vegetables in handling and food processing. For this purpose, indentation of tomato mesocarp cells with an atomic force microscope was used. The Young's modulus of a cell using the Hertz and Sneddon models, and stiffness were calculated from force-indentation curves. Use of two probes of distinct radius of curvature (20 nm and 10,000nm) showed that the measured elastic properties were significantly affected by tip geometry. The Young's modulus was about 100 kPa ± 35 kPa and 20 kPa ± 14 kPa for the sharper tip and a bead tip, respectively. Moreover, large variability regarding elastic properties (>100%) among cells sampled from the same region in the fruit was observed. We showed that AFM provides the possibility of combining nano-mechanical properties with topography imaging, which could be very useful for the study of structure-related properties of fruits and vegetables at the cellular and sub-cellular scale.

Fig. 3. Xylem morphology mutants affect electrical signals and deformation kinetics in leaf 13. Comparison of force changes and associated SPs (solid lines) for WT, irx3, and irx5 mutants. Experimental setup as in Fig. 2A. P. brassicae larvae were allowed to feed on leaf 8, and force changes and surface potential were measured on leaf 13. Both parameters were measured at 33 Hz. Individual traces were averaged according to the first SP depolarization minimum. For irx mutants, mean (solid lines) and SD envelopes (shaded area limited by dashed lines) were from at least 11 independent pairs of measurements. Asterisks indicate depolarization spikes in irx mutants.Kurenda et al.

Reports concerning the function of glutamate (Glu) in the electrical and movement phenomena in plants are scarce. Using the method of extracellular measurement, we recorded electrical potential changes in the stem of 3-week-old Helianthus annuus L. plants after injection of Glu solution. Simultaneously, circumnutation movements of the stem were measured with the use of time-lapse images. Injection of Glu solution at millimolar (200, 50, 5 mM) concentrations in the basal part of the stem evoked a series of action potentials (APs). The APs appeared in the site of injection and in different parts of the stem and were propagated acropetally and/or basipetally along the stem. Glu injection also resulted in a transient, approximately 5-h-long decrease in the stem circumnutation rate. The APs initiated and propagating in the sunflower stem after Glu injection testify the existence of a Glu perception system in vascular plants and suggest its involvement in electrical, long-distance signaling. Our experiments also demonstrated that Glu is a factor affecting circumnutation movements.

In this study, the biospeckle technique was evaluated for monitoring of apple bull’s eye rot development and product quality in general, during storage under various conditions and during subsequent shelf life. This non-destructive optical method is based on the analysis of laser light variations scattered from the sample. Apples of the cultivars ‘Pinova’ and ‘Topaz’, susceptible to bull’s eye rot, were used in two independent experiments. In the first, apples were non-destructively monitored for five months during cold storage. After that time, 34% of ‘Pinova’ and 21% of ‘Topaz’ apples displayed visible surface lesions. The increase of biospeckle activity was observed during the development of fungal disease. In the second experiment various storage conditions were used and apples were tested during their shelf life by non-destructive and destructive methods. This study showed that biospeckle activity decreased during shelf life, irrespective of storage conditions.