GLR-dependent calcium and electrical signals are not coupled to systemic, oxylipin-based wound-induced gene expression in Marchantia polymorpha.

In angiosperms, wound-derived signals travel through the vasculature to systemically activate defence responses throughout the plant. In Arabidopsis thaliana, activity of vasculature-specific Clade 3 glutamate receptor-like (GLR) channels is required for the transmission of electrical signals and cytosolic Ca2+ ([Ca2+]cyt) waves from wounded leaves to distal tissues, triggering activation of oxylipin-dependent defences. Whether nonvascular plants mount systemic responses upon wounding remains unknown. To explore the evolution of systemic defence responses, we investigated electrical and calcium signalling in the nonvascular plant Marchantia polymorpha. We found that electrical signals and [Ca2+]cyt waves are generated in response to mechanical wounding and propagated to nondamaged distal tissues in M. polymorpha. Functional analysis of MpGLR, the only GLR encoded in the genome of M. polymorpha, indicates that its activity is necessary for the systemic transmission of wound-induced electrical signals and [Ca2+]cyt waves, similar to vascular plants. However, spread of these signals is neither coupled to systemic accumulation of oxylipins nor to a transcriptional defence response in the distal tissues of wounded M. polymorpha plants. Our results suggest that lack of vasculature prevents translocation of additional signalling factors that, together with electrical signals and [Ca2+]cyt waves, contribute to systemic activation of defences in tracheophytes.

Mechanosensation in leaf veins.

Whether the plant vasculature has the capacity to sense touch is unknown. We developed a quantitative assay to investigate touch-response electrical signals in the leaves and veins of Arabidopsis thaliana. Mechanostimulated electrical signaling in leaves displayed strong diel regulation. Signals of full amplitude could be generated by repeated stimulation at the same site after approximately 90 minutes. However, the signals showed intermediate amplitudes when repeatedly stimulated in shorter timeframes. Using intracellular electrodes, we detected touch-response membrane depolarizations in the phloem. On the basis of this, we mutated multiple Arabidopsis H+-ATPase (AHA) genes expressed in companion cells. We found that aha1 aha3 double mutants attenuated touch-responses, and this was coupled to growth rate reduction. Moreover, propagating membrane depolarizations could be triggered by mechanostimulating the exposed primary vasculature of wild-type plants but not of aha1 aha3 mutants. Primary veins have autonomous mechanosensory properties which depend on P-type proton pumps.

Insect-damaged Arabidopsis moves like wounded Mimosa pudica.

Slow wave potentials (SWPs) are damage-induced electrical signals which, based on experiments in which organs are burned, have been linked to rapid increases in leaf or stem thickness. The possibility that pressure surges in injured xylem underlie these events has been evoked frequently. We sought evidence for insect feeding-induced positive pressure changes in the petioles of Arabidopsis thaliana Instead, we found that petiole surfaces of leaves distal to insect-feeding sites subsided. We also found that insect damage induced longer-duration downward leaf movements in undamaged leaves. The transient petiole deformations were contemporary with and dependent on the SWP. We then investigated if mutants that affect the xylem, which has been implicated in SWP transmission, might modify SWP architecture. irregular xylem mutants strongly affected SWP velocity and kinetics and, in parallel, restructured insect damage-induced petiole deformations. Together, with force change measurements on the primary vein, the results suggest that extravascular water fluxes accompany the SWP. Moreover, petiole deformations in Arabidopsis mimic parts of the spectacular distal leaf collapse phase seen in wounded Mimosa pudica We genetically link electrical signals to organ movement and deformation and suggest an evolutionary origin of the large leaf movements seen in wounded Mimosa.

Single-cell damage elicits regional, nematode-restricting ethylene responses in roots.

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 Ca2+ 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.

Identification of cell populations necessary for leaf-to-leaf electrical signaling in a wounded plant.

The identity of the cell files necessary for the leaf-to-leaf transmission of wound signals plants has been debated for decades. In Arabidopsis, wounding initiates the glutamate receptor-like (GLR)-dependent propagation of membrane depolarizations that lead to defense gene activation. Using a vein extraction procedure we found pools of GLR-fusion proteins in endomembranes in phloem sieve elements and/or in xylem contact cells. Strikingly, only double mutants that eliminated GLRs from both of these spatially separated cell types strongly attenuated leaf-to-leaf electrical signaling. glr3.3 mutants were also compromised in their defense against herbivores. Since wounding is known to cause increases in cytosolic calcium, we monitored electrical signals and Ca2+ transients simultaneously. This revealed that wound-induced membrane depolarizations in the wild-type preceded cytosolic Ca2+ maxima. The axial and radial distributions of calcium fluxes were differentially affected in each glr mutant. Resolving a debate over which cell types are necessary for electrical signaling between leaves, we show that phloem sieve elements and xylem contact cells function together in this process.

Determination of the Optimum Harvest Window for Apples Using the Non-Destructive Biospeckle Method.

Determination of the optimum harvest window plays a key role in the agro-food chain as the quality of fruit depends on the right harvesting time and appropriate storage conditions during the postharvest period. Usually, indices based on destructive measurements are used for this purpose, like the De Jager Index (PFW-1), FARS index and the most popular Streif Index. In this study, we proposed a biospeckle method for the evaluation of the optimum harvest window (OHW) of the "Ligol" and "Szampion" apple cultivars. The experiment involved eight different maturity stages, of which four were followed by long cold storage and shelf life to assist the determination of the optimum harvest window. The biospeckle activity was studied in relation to standard quality attributes (firmness, acidity, starch, soluble solids content, Streif Index) and physiological parameters (respiration and ethylene emission) of both apple cultivars. Changes of biospeckle activity (BA) over time showed moderate relationships with biochemical changes during apple maturation and ripening. The harvest date suggested by the Streif Index and postharvest quality indicators matched with characteristic decrease in BA. The ability of biospeckle method to characterize the biological state of apples was confirmed by significant correlations of BA with firmness, starch index, total soluble solids and Streif Index, as well as good match with changes in carbon dioxide and ethylene emission. However, it should be noted that correlations between variables changing over time are not as meaningful as independent observations. Also, it is a well-known property of the Pearson's correlation that its value is highly susceptible to outlier data. Due to its non-selective nature the BA reflected only the current biological state of the fruit and could be affected by many other factors. The investigations showed that the optimum harvest window for apples was indicated by the characteristic drop of BA during pre-harvest development. Despite this, at the current state of development the BA method cannot be used as an indicator alone. Due to rather poor results for prediction in OHW the BA measurements should be supported by other destructive methods to compensate its low selectivity.

Axial and Radial Oxylipin Transport.

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.

Electrical potential oscillations--movement relations in circumnutating sunflower stem and effect of ion channel and proton pump inhibitors on circumnutation.

The physiological control and molecular mechanism of circumnutation (CN) has not yet been fully understood. To gain information on the CN mechanism, the relationship between the changes of electrical potential and movement in the circumnutating sunflower stem and effect of ion channels and proton pump inhibitors on CN parameters were evaluated. Long-term electrophysiological measurements and injection of solutions of ion channel inhibitors (ICI) into sunflower stem with the simultaneous time-lapse recording of the movement were made. The oscillations of electrical potential (OEP) - movement relations - consist of cells depolarization on the deflected side of the stem and, at this same time, cells hyperpolarization on the opposite side of the stem. The delay of organ movement in relation to electrical changes of approximately 28 min (22% of the period) may indicate that the ionic fluxes causing the OEP are the primary phenomenon. The biggest decrease of CN period was observed after injection of proton pump (approximately 26%) and cation channel (approximately 25%) inhibitors, while length and amplitude were reduced mainly by calcium channel inhibitors (approximately 67%). Existence of OEP only in circumnutating part of sunflower stem and reduction of CN parameters and OEP amplitude after application of ICI prove that the CN cellular mechanism is associated with transmembrane ion transport.

Effect of Cytochalasin B, Lantrunculin B, Colchicine, Cycloheximid, Dimethyl Sulfoxide and Ion Channel Inhibitors on Biospeckle Activity in Apple Tissue.

The biospeckle phenomenon is used for non-destructive monitoring the quality of fresh fruits and vegetables, but in the case of plant tissues there is a lack of experimentally confirmed information about the biological origin of the biospeckle activity (BA). As a main sources of BA, processes associated with the movement inside the cell, such as cytoplasmic streaming, organelle movement and intra- and extracellular transport mechanisms, are considered. The aim of this study is to investigate the effect of metabolism inhibitors, connected with intracellular movement such as cytochalasin B, lantrunculin B, colchicine, cycloheximid, dimethyl sulfoxide (DMSO) and mixture of ion channel inhibitors, injected into apples, on BA. Two methods of BA analysis based on cross-correlation coefficient and Laser Speckle Contrast Analysis (LASCA) were used. DMSO, lantrunculin B and mixture of ion channel inhibitors have a significant effect on BA, and approximately 74 % of BA of apple tissue is potentially caused by biological processes. Results indicate that the functioning of actin microfilaments and ion channels significantly affect BA.

Determination of the elastic properties of tomato fruit cells with an atomic force microscope.

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 10,000 nm) 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.

Application of the biospeckle method for monitoring bull's eye rot development and quality changes of apples subjected to various storage methods-preliminary studies.

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.

Glutamate induces series of action potentials and a decrease in circumnutation rate in Helianthus annuus.

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.