Papel de los genes "Calcineurin b-like protein 10 (SLCBL10)" y "Respiratory burst oxidase g (SlRBOHG)" en la respuesta de tomate a estreses abióticos

Abstract

Climate change has dramatic consequences on agriculture worldwide because of the intensified impact of multiple abiotic stresses on crops. In Europe, the Mediterranean area is the most affected by climate change, where frequency, intensity and duration of stresses such as drought, high temperatures and salinity have been increasing, and in the particular case of the last one because of use by farmers of water with high levels of salts due to scarcity of good quality water sources. Given the negative effects of these stresses on the productivity of crops of such agro-economic importance as tomato (Solanum lycopersicum), which is the horticultural species supplying most of the functional compounds to the human diet due to its so high per capita consumption, and considering the predictions of a rising world population driving an increase in food demand, a priority research objective in agrarian science is to advance in the knowledge of the mechanisms and genes involved in tolerance to abiotic stresses, particularly in crops of such agronomic interest as it is tomato. The objective of this thesis is to advance in the knowledge of the role of two key tomato genes involved in tolerance to abiotic stresses throughout the plant development cycle: CALCINEURIN B-LIKE PROTEIN CBL10 (SlCBL10) and RESPIRATORY BURST OXIDASE G (SIRBOHG). To achieve these objectives, phenotypic and agronomic analyses of plants grown under different experimental conditions have been combined, including controlled, semi-controlled and uncontrolled growth conditions, as well as analyses fulfilled at anatomical, physiological and molecular levels. Regarding the study on SlCBL10 gene, it has been verified that it plays a very important role in reproductive development of tomato, since the knockout mutation of this gene significantly reduces fruit yield when plants are grown under optimal conditions. In addition, it is also involved in the incidence of Blossom End Rot (BER). Effects of knockout mutation or silencing of the gene are very much related to alterations in Ca2+ homeostasis, since it is observed a reduction in Ca2+ transport to sink organs that required this nutrient for their development. A very interesting characteristic of SlCBL10 is its opposite role in shoot and root when the plant is grown in saline conditions. Thus, disruption or silencing of the gene induces hypersensitivity to salt stress in the shoot and therefore it is a positive regulator of tolerance in this part of the plant, while it is a negative regulator of such tolerance in the root since plant material with loss-of-function of the gene confers salt tolerance when used as a rootstock. Interestingly, the rootstock carrying the silencing SlCBL10 gene are capable of increasing fruit yield of untransformed plant (WT) used as scion, and salinity tolerance of grafted plants is related to changes in Na+ and K+ plant homeostasis, and of expression of the main genes encoding transporters of both cations. Furthermore, the first results on the role of SlCBL10 in tomato tolerance to other abiotic stresses are presented, such as drought and high temperatures, and they hint at the fact that show that SlCBL10 overexpression induces sensitivity to heat stress, contrary to the response observed for salt stress. These results have a great interest to advance in the knowledge about the predominant mechanisms triggered in the plant response to a combination of abiotic stresses, which has a key importance given the 26 multiple abiotic stresses that affect crop growth and production in Mediterranean environmental conditions. Advances in the knowledge about the role of the SlRBOHG tomato gene, involved in the production of reactive oxygen species (ROS), have been carried out via the characterization of two mutants showing inhibition of the gene expression, called sodgat1-2 (sodium gatherer 1-2). These two mutants are characterized by the massive transport of Na+ from the roots to the shoot when grown in saline conditions. The knockout mutation of SlRBOHG causes a high sensitivity to salinity associated with a high transport of water and Na+ from root to shoot, a response observed both in non-transpiration (in vitro) and transpiration (in vivo) conditions. This physiological alteration is mainly associated with changes in the expression of ion transporters in mutant roots, in particular with the reduction of the expression of SlHKT1;2, one of the most important genes involved in regulation of Na+ homeostasis in tomato. This response depends on the production of H2O2 under saline conditions, since the sodgat1-2 mutants do not accumulate it with salinity, contrary to WT plants. Interestingly, the high sensitivity to salinity caused by disruption of the SlRBOHG is maintained throughout the plant cycle and in different environmental conditions, which hints at SlRBOHG as a key gene in vegetative development and, especially, in reproductive development, where loss-of-function causes major production losses. In addition, for the first time it is shown that the main effect caused by disruption of SlRBOHG at the reproductive level is a high degree of flower abscission induced by salinity, where important changes occur in the abscission zone (AZ) of the flower pedicel. Among the processes affected in the induced abscission under salinity, we have observed hormonal alterations such as reduced levels of auxin and ABA in leaves and flowers of the mutants and alterations in the levels of primary metabolites, especially sugars that experience a reduction of their transport from the source leaf towards sink flower and fruit. But abscission seems to be mainly linked to high transport of Na+ to the AZ in the mutants, since the accumulation of this cation in the AZ is even higher than the values detected in the flowers. These results will serve as a basis to advance in the identification of the key processes involved in premature abscission of reproductive organs, since flower abscission is an agronomic trait of critical importance to maintain production in climate change conditions. Finally, simultaneous co-expression tests of SlCBL10 and SlRBOHG genes with different CIPKs in HEK cells cultures showed that the SlCBL10-SlCIPK23 module is capable of activating SlRBOHG, leading to a significant increase in ROS in the presence of Ca2+. This last result evidence that these two genes, objectives of study in this PhD thesis, SlCBL10 and SlRBOHG, may have a key joint function in the crossover dialogue between Ca2+ and ROS for stress signalling in tomato Autor/es principal/es: Estrada Fortes, Yanira