CE20 - Biologie des animaux, des organismes photosynthétiques et des microorganismes

Abscisic acid-mediated control of K+ transport in the grape berry – KABAGRAPE

Abscisic acid-mediated control of K+ transport in the grape berry

The project aims to identify regulatory pathways that, in response to high temperatures linked to climate change, cause excessive accumulation of potassium (K+ ion) in grape berries. This accumulation is deleterious for the quality of the wines. We are interested in pathways that depend on abscisic acid stress hormone (ABA) and that lead to the regulation of the activity of K+ transport systems in the berry.

Identification of regulatory networks and determination of the function of the different partners

The project is divided into four workpackages to identify and validate the regulatory networks, and then to understand in more detail the function of each protein within the network.<br />WP1 (partner 1): identification of proteins involved in the regulation of potassium transport systems in the grape berry.<br />WP2 (partner 2): functional analysis of transport systems and their regulators, genetic validation by translational biology in Arabidopsis thaliana.<br />WP3 (partners 1 and 2): Analysis of gene expression: localization and dependence on stress of high temperatures.<br />WP4 (partner 1): Functional effect of the validated partners on the activity of transport systems in different heterologous systems (yeast, xenopus oocyte, plant tissues) and in grape berry protoplasts.<br />The ultimate goal is to give molecular tools for the selection, within a grape variety, of lines that accumulate less potassium in the berry.

Several approaches are proposed, both to identify regulators of transport systems by protein-protein interactions and to understand the role of these regulators.
The identification of candidate proteins is carried out by protein-protein interaction screenings in yeast (two-hybrid and split-ubiquitin). As no corresponding cDNA library has been produced for the grape berry, we propose to create two-hybrid and split-ubiquitin libraries as part of the project.
As the vine is difficult to undergo genetic analysis, genetic validation will be carried out in the model species Arabidopsis thaliana, and more particularly in germinating seeds which are well studied for ABA signalling. Mutants for homologous/orthologous genes encoding channels/transporters and their regulators are analyzed. The phenotypes sought concern seed dormancy, ABA/gibberellic acid balance, resistance to ABA, growth on a low-K+ medium... Epistatic interactions will be analyzed by the associations of mutations and by the creation of lineages overexpressing the genes of interest.
Gene expression studies will be performed by Q-PCR and in situ hybridization. Q-PCR measurements will be correlated with quantifications of ABA and its metabolites in the same tissues.
For functional studies, intended to estimate the impact of the regulatory protein on the activity of the target channel, co-expression in xenopus oocytes, followed by measurements of currents by the two-electrode voltage-clamp method, is the preferred technique.
In planta analyses will be carried out in the tobacco epidermis (co-expression of proteins with a fluorescent tag, to visualize their co-location and the effect of the presence of the regulatory protein on the subcellular localization of the channel or transporter) and transformed grape berry protoplasts (patch-clamp).

- For Workpackage 1 (partner 1), we have highlighted control networks starting from the ABA to arrive at the terminal effectors that constitute the potassium channels. These networks involve ABA receptors (PYR/PYL/RCAR), protein phosphatases 2C involved in ABA signaling, as well as kinases not yet listed as channel regulators. To identify new regulators, we produced grape berry RNAs in sufficient quantities to prepare cDNA librairies, and began creating a library for two-hybrid screenings.
- For workpackage 2, many mutants were genotyped for T-DNA insertions in genes encoding Arabidopsis' K+ transport systems. The results of the first phenotypings show that some lines have phenotypes that can be marked regarding germination or hormonal responses.

As soon as the results of the screenings for the interactions are available, the mutant lines corresponding to the genes of interest will be searched and phenotyped, and the effect of the candidate proteins on the activity of the channels will be measured by electrophysiology in xenopus oocytes.
The more distant perspectives are described in the «Main issues« and «Methods« sections.

No publication yet for this project.

KABAGRAPE aims to identify the abscisic acid (ABA)-dependent gene networks involved in the increase in potassium (K+) loading observed in response to high temperatures during grape ripening. Indeed, the onset of the maturation of the berry (called veraison) is triggered by ABA, and this hormone well known for its role in the response to water stress is also involved in the adaptation of the vine to high temperatures. However, in this period of climate change, it is known that the exposure of grapes to high temperatures is the external factor that most affects the quality of the berry at harvest, leading to the production of wines of low organoleptic quality with a reduced ageing potential. In particular, the increase in temperature favors the accumulation of K+ which, if it is found in excess in the berry, has harmful effects on the quality of the wine.
ABA has been identified as an essential player in grape ripening. In the grape berry, the concentration of ABA increases at the time of veraison, then during maturation in case of high temperatures. In Arabidopsis, it is established that ABA controls the activity of K+ transport systems (Shaker channels and KUP transporters) and therefore the loading of K+ in different tissues. This K+ transport activity is regulated by clade A protein phosphatases 2C (PP2C) and kinases involved in ABA signaling, such as Calcineurin B-like Interacting Protein Kinases (CIPK), and SNF1-related kinases (SnRK2), via direct protein-protein interactions.
In the grape berry, we want to understand how ABA regulates the entry (via the phloem) and the accumulation of K+ through its action on transport systems. In addition to the already published CIPKs, we have recently identified PP2Cs interacting with Shaker channels, and new candidate proteins belonging to families not yet associated with the transport of K+ in plants: a receptor-like kinase (RLK), which interacts with a PP2C of the ABA signaling pathway, and an ABC transporter close to Arabidopsis ABA transporters. In KABAGRAPE, we will analyze these new regulators and their close counterparts and look for other candidates.
The project is divided into four workpackages (WP 1 to 4). WP1 will aim to identify regulatory proteins interacting with the berry's K+ transport systems, both by systematic interaction tests and by screening of cDNA libraries in two-hybrid and split-ubiquitin. We selected as primary targets the most expressed Shaker channels in the berry, as well as a KUP transporter highly induced from veraison. To validate the ABA dependence of candidate proteins and their relationships with transport systems, we will use in WP2 an original strategy based on phenotyping at the germination stage of Arabidopsis mutants deficient for the orthologs of the genes of interest (coding the K+ transport systems and their regulators), of lines overexpressing these genes, and of combinations of genotypes. In WP3, we will characterize the spatiotemporal expression of the genes of interest in the grape berry and their response to stress, in relation to the accumulation of ABA and its derivatives. Finally, WP4 will aim to understand the functional role of regulatory proteins on K+ transport systems: effects on their subcellular localization and on K+ transport activity (by electrophysiology in Xenopus oocyte and on berry protoplasts, and/or yeast complementation). Our consortium combines 2 groups with complementary skills: (1) (electro) physiology and molecular biology of K+ transport in Arabidopsis and grapevine, (2) physiology of seed germination and hormonal regulations.

Project coordination

Isabelle Chérel (Biochimie et Physiologie Moléculaire des Plantes)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

IJPB Institut Jean-Pierre BOURGIN
BPMP Biochimie et Physiologie Moléculaire des Plantes

Help of the ANR 403,094 euros
Beginning and duration of the scientific project: December 2020 - 48 Months

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