Resumen de tesis. Análisis funcional de proteín-fosfatasas de tipo 2-C en la dormición/germinación y las respuestas a estrés en Arabidopsis Thaliana

Abstract

[EN] Plant seed is the result of ovule maturation and it is formed by an embryo surrounded by protective structures and storage tissues such as endosperm or perisperm, in those species which cotyledons do not accumulate reserves. Seed plays a key role in plant life cycle regarding its survival as a species. It is the dispersal unit of the plant, which is able to survive the period between seed maturation and the establishment of the next generation as a seedling after it has germinated. For this survival, the seed, mainly in a dry state, is well equipped to sustain extended periods of unfavourable conditions.

Germination is a process that begins with water uptake by the seed (imbibition) and ends with the emergence of the embryonic axis, usually the radicle, through the structures surrounding it. On the other hand, dormancy is the temporary failure of a seed to complete germination under favourable conditions and it allows for the dispersal of seeds in space and time.

To optimise germination over time, the seed enters in a dormant state. Dormancy prevents pre-harvest germination as well. Numerous studies have been performed to better understand how germination is controlled by various environmental factors and plant hormone. Nevertheless, many aspects about the process of germination are still unknown.

The transition from dormancy to germination depends on several factors that affect seeds simultaneously, in Arabidopsis it requires the removal of several “blocks” to germination, such as after-ripening, chilling and exposure to nitrate and light, but the termination of dormancy and the triggering of germination is not due to only one factor but several, that generate a cross talk between them and hormonal regulation and provide an integrated network that leads to the the decision of germinate or stay dormant depending on ecological opportunities.

Hormonal regulation is due to abscisic acid (ABA) and gibberellins (GA), the first one has an inhibitory and the second one a promotive effect in the induction of germination.

Genetic evidence of this antagonistic effect was provided by molecular genetics through the isolation of ABA-deficient mutations as suppressors of non-germination due to GA deficiency and the GA response mutant sleepy as suppressor of abi11-1. Dormancy maintenance is due to de novo synthesis of ABA during imbibition. ABA is produced via carotenoid pathway and NCED is the enzyme that catalyzes the limiting step of the synthesis: the conversion of 9´-cis-neoxanthin and 9-cis-violaxanthin to xanthoxin by 9-cis-epoxycarotenoid dioxygenase. It regulates the rate of ABA production, associated with the induction and maintenance of dormancy.

Release from dormancy, on the other hand, is correlated with ABA catabolism by an other key hormone, CYP707A2 and also to increased GA synthesis of GA3ox1, GA3ox2 and decreased expression of inactivating enzyme GA2ox.

But the relative activities of ABA and GA not only depend on its quantity but also on the perception of hormones by its receptors and subsequent signalling transduction pathways, that eventually influence gene expression. Therefore, the identification of hormone transduction pathways is critical to unravelling these processes where posttranslational modifications of signalling proteins by phosphorylation or dephosphorylation, and ubiquitination, which affects their stability, play a critical role.

Phosphorylation is one of the major reversible signal transduction control mechanisms, mediated by protein kinases and protein phosphatases. PP2Cs are universally distributed group of phosphatases, forming the largest phosphatase family in plants. It is divided in ten groups from A to J. Members of PP2Cs from cluster A and D are the main characters of this dissertation.

Cluster A PP2Cs (A-PP2Cs), known for years as major negative regulators of ABA signalling, have been revealed as components of the its receptor complex. In conditions of non-ABA, PP2Cs continuously dephosphorylate SnRKs, blocking the transduction response to ABA. When ABA is present, PYR/PYL/RCAR proteins, ABA receptors, are able to bound to ABA and thereby stably interact with PP2Cs and inhibit its dephosphorylation by disabling its catalytic function.

On the other hand, cluster D PP2Cs (D-PP2Cs), largely unknown have been recently related with elongation processes.

In the first chapter of this dissertation, A-PP2Cs and ABA receptors, PYR/PYL/RCAR proteins, are characterized in oxidative stress responses in germination. To accomplish it, germination assays in the presence of methyl-viologen, a reactive oxygen species generator, are performed in the presence of several mutant lines (with overexpression, knock-out or hipermorphic phenotypes) of A-PP2Cs and PYR/PYL/RCAR. Also, the activity of the most important enzymes of the plant antioxidant system, superoxide dismutase, catalase and glutathione reductase, are measured in seeds of all these lines to further unravel the function of both types of proteins.

In the second part, a Yeast Two Hybrid (Y2H) is performed to find D-PP2Cs interacting proteins. The interactions are fully characterized by in planta verification with Bimolecular complementation assays (BiFC). A triple D-PP2C phosphatase mutant is generated and characterized in germination assays. Multiple hormone assays are performed as well to elucidate the relationship between D-PP2Cs and hormones signalling pathway. One of the D-PP2Cs interacting proteins is also characterized in the same conditions, showing the clear effect of the interaction in biological function.