Final Report Summary - WOUND IN EARTH (Wound induced electrical signals in Arabidopsis thaliana) The ability to produce long-distance electrical signals is an advantageous trait that allows multi-cellular organisms reacting as a whole to external stimuli. This trait has evolved independently in plants and animals, and thus represents a case of convergent evolution. The ultra-fast, long distance electrical signals in animal nervous systems are well studied using minimally invasive technology. In contrast, the electrical transmission in plants is a neglected area of research. Although plants respond to harsh stimuli by generating long-distance electrical signals, it is not known whether more realistic challenges, such as bites by herbivorous insects, could also induce these signals. Another problem is that the technology for acquisition of electrical signals in plants is highly invasive. Whereas the biophysics, molecular bases, and roles of long distance electrical signals in animals are well established, they are just beginning to be understood in plants. The major objective of the IIF-MC project WOUND IN EARTH was to qualitatively and quantitatively characterize the biophysical features and molecular bases of stimulus-induced electrical signals in the phloem of the model plant Arabidopsis thaliana. The main cells of the phloem, the sieve elements (SEs), are elongated cells connected end-to-end to form a continuous, low-resistant sieve tube system that spreads throughout the plant. For this reason, the phloem has been postulated as a major tissue for long-distance electrical transmission. However, the current techniques for studying the electrical properties of SEs are either too invasive, or too low-throughput to allow for quantitative studies. For this project, Dr. Vicenta Salvador-Recatalà conceptualized an alternative approach to acquire intracellular electrophysiological recordings from SEs. In this method, long distance electrical signals are acquired exclusively from SEs by means of living aphids (or other phloem-feeding hemipteran insects) that are integrated into the electrical penetration graph (EPG) circuit (more details can be found in the open-access article: http://onlinelibrary.wiley.com/doi/10.1111/nph.12807/full). Using these "EPG-electrodes" VSR obtained highly reproducible results that demonstrate that natural wounds inflicted by feeding caterpillars consistently trigger electrical signals in SEs in the vicinity of the wound (i.e. in the wounded leaf), as well as in SEs of unwounded leaves. Whereas the electrical signals in SEs near the wound consists of relatively fast, action potential-like depolarizations, the SEs in unwounded leaves can produce two different types of wound-induced electrical signals, depending on whether the wound is inflicted to a neighbor leaf or to a non-neighbor leaf. If in a neighbor's leaf, a SE produces two depolarizations: a first slow depolarization that requires, on average, fourteen seconds to fully activate, and a superimposed, action potential-like transient depolarization that requires only approximately one second to fully activate. If in a non-neighbor's leaf, a SE responds to distal damage with the slow depolarization only. Interestingly, leaves the SEs of which produce action potential-like signals in response to damage, also activate jasmonic acid-dependent intracellular signaling, which is functionally linked to expression of important defense genes. The results of this project provide evidence that the long-distance electrical signals transmitted through the phloem vascular system of plants are commonplace in nature, as well as for a functional link between these electrical signals and the induction of defensive biochemical/genetic responses of plants to damage. In neurons, electrical signals are produced by ions that flow in and out of the cell. These ionic currents change the concentration of ions inside the cell, in other words they create electrical waves. Specific proteins expressed in the plasma membrane of all cells, called ion channels, control the flow of these ions. Some ion channels open, to allow the ion flow, in response to a change in the membrane voltage. Others open when they bind a specific molecule, or ligand. The data from this project show that the ligand-gated, glutamate receptor-like (GLR) channels GLR3.3 and GLR3.6 which putatively mediate the influx of calcium into plant cells, are important molecular determinants of the long-distance electrical signals in the phloem of the model plant Arabidopsis. Future research with EPG-electrodes has the potential to make major contributions to the neglected field of stimulus-induced electrical signals in plants. Specifically, by identifying the full set of the molecular determinants of these electrical signals, and by establishing the functional connections between the plant electrical signals and their biochemical and genetic responses to external stimuli. Given the versatility of EPG-electrodes, they can be implemented on a wide variety of plants, including species of agricultural interest (rice, wheat, etc.). Characterising the electrical features of the sieve elements is necessary for a comprehensive understanding of the physiology of the phloem. Since the phloem is a major contributor to the production of plant materials that are important to humans, this knowledge may lead to devising ways to increase crop yields in order to meet the global needs for food and fuel.
