Final Report Summary - PLANTELEXIGNAL (Disclosing the molecular bases of electrical signalling in plants) Scientific contextPlants make use of different signalling pathways in order to co-ordinate their adapted physiological responses to physical, nutritional or biotic environmental constraints. Amongst these signalling pathways, the electrical one is thought to be the most convenient for rapid information transfer over long distances, e.g. action potentials (APs) propagated from roots to shoots. The pathways for propagation of APs (or AP-like signals) are mainly through symplasmic tissues such as the mesophyll in leaves and cortex in roots, or vascular tissues within the whole plant. With the completion of Arabidopsis and other plant genome sequence, it is now necessary to consider the most integrative aspects of plant physiology. In this context, the present research project has resulted from evidence that a number of cloned ion transport systems that have so far only been considered for their role in mineral nutrition, now appear to be worth considering also as possible components of the electrical signalling system in plants. Despite their importance, huge questions remain about the mechanisms of electrical signalling. Although there is a well-accepted hypothesis for the generation and propagation of plant APs, which involves the cellular influx of calcium and efflux of chloride and potassium through ion channels, little is known about the molecular identify or the regulation of these ion channels. This is despite the rapidly increasing knowledge concerning ion transport mechanisms in model plant species such as Arabidopsis thaliana. Project aims / mobilised resources Focusing on APs in the model plant species Arabidopsis thaliana, this project aimed to fill the gap between, first, the many reports on APs propagating in plants upon a broad range of stimuli/stresses (biotic or abiotic) and, second, the fairly large body of knowledge of the molecular properties of voltage-dependent ion channels likely to be involved in both AP generation and propagation. The overall project aim was, combining in vivo experimentation and computer-assisted mathematical modelling, to provide a model describing the molecular mechanisms underlying the generation and the propagation of electrical signals in plant tissues and their integration with Ca2+ signalling. To achieve this, ion transport mechanisms and electrical signalling in Arabidopsis were brought together. The involvement and the regulation of the ion channels involved in mediating ion fluxes during an AP were to be intensively investigated, with the final aim to result in a mathematical model of electrical signalling in plants. The project was managed, in Montpellier-France, by the group hosting the Marie-Curie IRG fellow (a returning European electrophysiologist with expertise in ion transport); this group having expertise in molecular electrophysiology of plant ion channels and having started (i) a computer-assisted mathematical modelling of plant biological processes (a confirmed post-doctoral fellow, appointed by "Agropolis Fondation" from June 1st 2009 to May 31th 2012, was in charge of this modelling), (ii) development of a new Ca2+ probe to image Ca2+ signalling in whole plants (including mature leaves), and (iii) study of Ca2+-dependent regulation of Shaker channels involved in electrical signalling (a PhD project, dedicated to this particular topic was started just when the IRG fellow joined the hosting group). The project was accomplished in connection with groups with expertise in (i) electrophysiology, especially in relation to electrical signalling (in Geneva/Switzerland and in Lublin/Poland) and (ii) computer-assisted mathematical modelling of plant biological processes (in Ottawa, Canada, in Potsdam, Germany, and in Bern, Switzerland). The project made use of the plant electrophysiology platform (managed by the Project coordinator, more information at http://www1.montpellier.inra.fr/ibip/bpmp/english/ressources/electrophysiology/EHEVflyerJuly2013.pdf) available in the hosting lab.Performed workWork during the three years of this project has consisted in (i) implementing in the host lab the experimental devices requested for recording electrical signals in Arabidopsis, (ii) recording these signals (similar to action potentials, denoted below "AP-like signals") in different Arabidopsis genotypes, (iii) implementing cutting-edge methods to image Ca2+ events in Arabidopsis aerial parts, (iv) recording Ca2+-signals in same experimental conditions as AP-like signals, (v) identifying Ca2+-dependent regulation mechanisms of ion channels involved in AP-like signals (the aim being to get insights into cross-talk between electrical signalling and calcium signalling), (vi) developing a mathematical model (computer-assisted modelling) of plant cell "excitability" and of ability of plant symplasms to propagate AP-like signals.Main achieved resultsWe have obtained a set of experimental results evidencing a role of two K+ channels in triggering, shaping and propagating AP-like electrical signals in Arabidopsis mature leaves. Briefly, one of these channels, AKT2, has an impact on "excitability", i.e. on the probability of AP-like elicitation after a mechanical or an electrical stimulation of a given strength. Knocking-out the AKT2 gene reduces significantly excitability. Conversely, expressing a gain-of-function mutant AKT2 gene (encoding an "hyperactive" version of the AKT2 channel) increases much excitability. It is noteworthy that comparative studies of plants expressing the same different AKT2 alleles (wild-type, KO/loss- or gain-of-function) have evidenced a role of AKT2 in sucrose (re-)loading of phloem cells through a control of membrane potential of these cells, where AP propagation is likely to take place (two publications, including one co-authored by the IRG fellow, in collaboration with partners in Potsdam, Germany). Besides, knocking-out GORK, another K+ channel-encoding gene, dramatically increases both the amplitude and the duration of AP-like electrical signals. This is in accordance with predictions of the mathematical model for plant cell excitability that we have implemented. A manuscript reporting these results about AKT2 and GORK role in excitability and features of AP-like signals, first-authored by the IRG fellow, has been prepared but not submitted yet (an issue addressed in Section 8 of this report). In all the genotypes we tested so far, however, the speed of AP-like propagation within leaf tissues shows, however, little variations in the 1 mm.s-1 range, and seems rather independent of the expression level of both AKT2 and GORK genes. This has been confirmed by independent experiments performed by our partners in Geneva and Lublin. Thus far, the mechanism of AP-like propagation remains unclear. In particular, it is uncertain, based on the available mathematical model of electrical properties of a plant symplasm, that this propagation results from cell-to-cell electro-coupling as we initially assumed. Interestingly, it is now known that "waves" of reactive oxygen species (ROS, signaling molecules) waves propagate through leaves at comparable speed suggesting a possible link with AP-like signals. Regarding the Ca2+ signalling, we obtained very promising results. First, development of the method for imaging Ca2+ events in leaves has been achieved. We are now able to record such Ca2+ events upon stress/stimuli similar to those used for AP recordings. Second, an much interestingly, these signals take place in the same leaf territories as APs, e.g. mesophyll patches and vein network in leaves. They often feature characteristics of "waves" with, in particular, a speed of propagation in the same range as the AP-like signals. This suggests a link between electrical and calcium signalling as predicted by the model of plant cell excitability developed in the group. Finally, we identified several Ca2+-dependent kinases (controlled by Ca2+ both transcriptionally and post-translationally) which target voltage-dependent K+ channels potentially involved in electrical signaling. Potential impactIt is the first time that ion channels identified at the molecular level are evidenced to underlie AP-like signal in a plant (here, Arabidopsis). This could make a breakthrough in physiology of stress signalling by plants, which is a trendy topic in plant science. The mathematical modelling incepted in the framework of this project will potentially offer insights into the mechanism of AP elicitation and propagation in plant tissues. The new method developed for real-time imaging of Ca2+ signalling in photosynthetic plant tissues is extremely promising. Five-fold gain in time resolution is obtained with the BRET-based Ca2+-probe that we introduced in Arabidopsis, which opens exciting perspectives (a methodological paper under revision). Thanks to this new method, Ca2+ events elicited by stimuli/stresses identical to those used to make AP-like signals could be recorded. That these "Ca2+ waves" do travel through plant leaves at similar speed as AP-like signals is also very interesting. This, indeed, suggests a link (eventually involving Ca2+-dependent kinases) between the two signalling pathways and offers clues to both a triggering mechanism of AP-like signals and a role for these electrical events, which are known for long but as yet poorly understood.
