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Novel insights into the sensing of salt stress in plants: understanding the relationship between salt stress response and cytosolic pH changes.

Periodic Reporting for period 1 - sigNal (Novel insights into the sensing of salt stress in plants: understanding the relationship between salt stress response and cytosolic pH changes.)

Período documentado: 2018-09-01 hasta 2020-08-31

Soil salinisation is a growing problem for agriculture worldwide. Evidence is available at the physiological and molecular levels that Na is the major cause of salt toxicity in most species. The Salt Overly Sensitive (SOS) pathway is one of the main regulatory systems responsible for Na homeostasis in plants. The SOS pathway is activated by salt stress and comprises three core components: SOS1, SOS2 and SOS3. SOS3 (CBL4) is a calcium (Ca) sensor with four Ca-binding sites (EF-Hand domains) that perceives the increase of intracellular Ca triggered by salt stress, recruits SOS2 (CIPK24) and the SOS2–SOS3 complex activates the downstream target protein SOS1 (NHX7). Cytosolic free Ca is a common second messenger in the signalling of a variety of abiotic stresses. Current thinking is that the specificity of Ca signalling is determined by the different patterns of cytosolic Ca level changes, often referred to as the “Ca signatures”, that are elicited by the stimulus. However, such a wide range of Ca-activated responses lead us to posit the existence of additional mechanisms relaying input signals that, together with this Ca signature, would initiate the specific response for a particular stress.

In this context, the overall aim of sigNal project was to gain novel insights into the early steps in the sensing and regulation of the salt stress response, for a better understanding of the salinity tolerance mechanisms of plants. In particular, I tried to show how salt stress signalling is discriminated from other biotic and abiotic stresses also leading to Ca spikes.

There are two main conclusions of this project: (1) salt stress promote an increase of cytosolic pH in the root tip, higher than the one observed under the same osmotic stress and high enough to be consider as having multiple cell consequences; (2) root tip works as a stress-sensing niche (SSN), were combination of stress-specific changes of pH and Ca, would be responsanble for the activation of stress-specific responses.
My research has focused on answering two general questions: (1) does salinity induce an alkaline pH shift in plant cells? (2) does SOS3 have pH-sensor function? To answer them I have used state-of-art sensing fluorescent indicators, confocal microscopy, GreenGate cloning system, Arabidopsis transgenic lines and yeast-based methodologies.

Unfortunately, it has been not possible to publish the results of sigNal project yet, but the manuscript is in progress and it will be submitted to an international peer-review journal as soon as possible. For this reason, a complete result overview cannot be publically publish here yet. However, in the attached figure, the pH heatmaps for different treatments are shown (A), where it can be obseved the site-restricted location of the root alkalinization. This zone has been used to define the MEZ ROI (B), that has been used to calculate actual pH values under diferent treatments. Attached figure also shows the actual pH values for 1 h incubation control, mannitol, KCl and NaCl treatments (C), where the difference in alkalinisation leves can be observed.

Regarding the dissemination of results, they were presented at scientific conferences in 2019: TNAM (Tri-National Arabidopsis Meeting) and SEB (Society for Experimental Biology) conference. sigNal project and results were presented as posters in both conferences and the best poster prize was awarded during TNAM. Unfortunately, ICAR (International Conference on Arabidopsis Research) and GRC (Gordon Research Conferences) 2020 were cancelled because the Corona crisis. sigNal project and its results were also presented at COS meetings and seminars: COS PhD-Postdoc seminars, Joint Lab Meeting and COS PhD-Postdoc Retreat.
The implementation of sigNal project clearly contribute to enhance our knowledge about the mechanisms by which plant roots translate changes in the media/soil to specific responses. In particular, it has revealed new insights about the signalling of salt stress in Arabidopsis roots. A better understanding of salinity tolerance mechanisms of plants will allow the development of crop varieties adapted to suboptimal environmental conditions and, then, results from sigNal project have potential interest for scientific experts in the field of plant signalling pathways.

The proposal is also relevant from a methodological point of view, since dual-sensing genetically encoded fluorescent indicator R-GECO1-E2GFP has been used to visualized in vivo and simultaneouslly changes in Ca and pH.