Project

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Molecular and structural characterisation of the vacuolar ALMT anion channels in Arabidopsis thaliana

Applicant De Angeli Alexis
Number 141090
Funding scheme Project funding
Research institution Institut des Sciences Végétales CNRS Bâtiment 23
Institution of higher education University of Zurich - ZH
Main discipline Biophysics
Start/End 01.05.2012 - 31.08.2015
Approved amount 213'848.00
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All Disciplines (2)

Discipline
Biophysics
Biochemistry

Keywords (6)

Arabidopsis thaliana; vacuole; transporter; malate; ion channel; chloride

Lay Summary (English)

Lead
Lay summary

Ion transport in plant is of central interest in plant biology. Indeed, ion fluxes across the membranes are essential for example in mineral nutrition, cell elongation, signal transduction and stomata movements. Therefore, understanding the molecular details of ion transport is a major issue in plant biology.  Different protein families are involved in mediating the transport of the different ion species. In the last decade several anion transporters families have been identified.   Among them the ALMT (Aluminium Activated Malate Channel) was shown to be implicated in  the transport of anions across the plasma membrane and the tonoplast. In the present project we will focus on the members of the ALMT family localised in the vacuolar membrane. We want to characterise their functional properties and to understand their role in the plant anion homeostasis. Several fundamental questions are still open concerning these proteins. Are the different vacuolar ALMTs involved in different cellular processes? Do they perform their function as homomers or heteromers? What is their role for plant growth, development and stress resistance? We will address these issues using different approaches:

A)    an electrophysiological characterisation of the vacuolar ALMTs for which any functional information is lacking .

B)     a combination of biochemical and microscopy techniques to investigate the quaternary structure of these proteins and to elucidate if they can form heteromeric complexes in vivo

C)    an analysis of single and multiple loss-of-function mutant plants and of multiple silenced lines that we will generate during this project.

The combination of these different approaches will allow us understanding the role of the vacuolar ALMTs at different level of integration: the organelle, the cell and the plant.

Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Cytosolic nucleotides block and regulate the Arabidopsis vacuolar anion channel AtALMT9.
Jingbo Zhang, Martinoia Enrico, De Angel Alexis (2014), Cytosolic nucleotides block and regulate the Arabidopsis vacuolar anion channel AtALMT9., in Journal of Biological Chemistry, 25581.
Identification of a probable pore-forming domain in the multimeric vacuolar anion channel AtALMT9
Zhang Jingbo, Baetz Ulrike, Kruegel Undine, Martinoia Enrico, De Angeli Alexis (2013), Identification of a probable pore-forming domain in the multimeric vacuolar anion channel AtALMT9, in Plant Physiology, 2 830-843.

Collaboration

Group / person Country
Types of collaboration
C.N.R. Italy (Europe)
- in-depth/constructive exchanges on approaches, methods or results

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Gordon Conference: Organellar Channels & Transporters Talk given at a conference A Malate-Activated Vacuolar Chloride Channel Required for Stomatal Opening in Arabidopsis 15.06.2015 Bentley, United States of America De Angeli Alexis;
International Symposium “Dynamic storage function plant vacuoles Talk given at a conference A Malate-Activated Vacuolar Chloride Channel Required for Stomatal Opening in Arabidopsis 11.05.2015 Heidelberg, Germany De Angeli Alexis;
The Enigmatic Chloride Ion: Transport, Regulation, and Roles in Physiology Talk given at a conference Function of Intracellular Anion Transporters during Stomata Movement 04.09.2013 WoodsHole, United States of America De Angeli Alexis;


Abstract

The aim of this project is to better understand the anion homeostasis in the model plant Arabidopsis thaliana. Anions play several roles in the plant. They can be essential nutrients or metabolites (e.g. nitrate, phosphate and malate) and act as osmolytes to maintain the turgor pressure. The vacuole stores and releases anions depending on the cell demand. Therefore it plays an essential role as a reserve for nutrients, to keep the cytosolic homeostasis and to maintain the cell turgecence. Consequently, the tonoplast transporters are of extreme interest to understand anion fluxes in plants. The functional characterisations of the anion transporters have been an important topic in the last decade. However, only recently the molecular identity of some anion transporters has been unveiled. The research group of prof. E. Martinoia (where DAA worked in the last 2 years) strongly contributed to the understanding and characterisation of the vacuolar anion transporters from the early functional characterisation to the more recent molecular identification. In this laboratory different kinds of anion transporters were identified, from the ATP dependent ABC transporters to the passive anion channels of the ALMT family. The first members of the ALMT family described were TaALMT1 and AtALMT1. It was found that these proteins are plasma membrane anion channels involved in the excretion of dicarboxylate from root epidermal cells. Subsequently, the group of prof. Martinoia found that AtLAMT9 and AtALMT6 are vacuolar malate channels expressed in the mesophyll and guard-cells, respectively. In his former work DAA identified and characterised the first vacuolar nitrate transporter, AtCLCa. Using a combination of electrophysiology, molecular modelling and in planta measurements it was possible to investigate the structural basis of the selectivity and the regulation of the transport activity by nucleotides. In prof Martinoia’s laboratory DAA contributed to the characterisation of AtALMT6, a calcium and pH regulated vacuolar malate channel. Currently DAA is working on AtLMT9 as a model to understand the structural organisation of ALMT channels and found that AtALMt9 can work as a chloride channel, the first identified in the vacuole. Therefore in this project we propose to i) investigate AtALMT4 and AtALMT5 which we know to be localized in the vacuolar membrane but which so far have not been characterized for their expression and function; ii) carry out experiments to learn more about the structure of functional ALMTs by investigating whether they form oligomers as suggested by our recent results and iii) to produce multiple knock-out plants, since no obvious phenotype was observed so far for single-gene knock-out plants. Due to its importance and impact on plant metabolism and growth, the cellular anion homeostasis is of particular interest but how its is achieved and regulated is still elusive. The ALMT family members are major actors of these processes and therefore studying them is an important issue in plant physiology. Moreover, ALMTs are a channel family exclusive to plants. Therefore, a biophysical characterisation is of particular interest since it could lead to unexpected and exiting results.
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