Caracterización funcional dela enzima de la pared celular xiloglucano endotransglucosilasa.

RESUMEN El objetivo general de este trabajo Tesis Doctoral se basó en la caracterización fisiológica, bioquímica y molecular de la enzima xiloglucano endotransglucosilasa/hidrolasa. Para ello, se utilizaron plantas de tomate (Solanum lycopersicum L.) de dos variedades y plantas transgénicas que tenían modificada la expresión de SlXTH1. De modo que estudiamos la participación de la XET en el crecimiento de plantas, elongación y expansión celular y su implicación en el crecimiento y ablandamiento de los frutos. Se evaluó el papel de las enzimas implicadas en el metabolismo de la pared celular de frutos durante la infección por un patógeno fúngico y en particular, de la XET. Además, estudiamos la posible regulación de las XTHs por etileno. Las conclusiones más relevantes fueron las siguientes: 1. Las dos modificaciones génicas realizadas en el gen SlXTH1 en Solanum lycopersicum L. provocaron un aumento y disminución de la expresión génica de este isoenzima que se correspondió, con el aumento y disminución de la actividad XET. Es la primera vez que se han descrito plantas transgénicas que tienen aumentados los niveles de expresión de SlXTH1 y muestran un aumento de la actividad XET. 2. En plántulas, la sobre-expresión de SlXTH1 provocó cambios irreversibles y permanentes en los enlaces del xiloglucano con el resto de los componentes, que posiblemente, fueron los responsables del aumento de la extensibilidad en la zona apical, y de la alteración del fenotipo. Esto sugiere que la XET está implicada en el crecimiento, y más concretamente, la isoenzima codificada por SlXTH1. 3. En las plantas que tienen disminuida la expresión de SlXTH1, se encontraron cambios en el fenotipo (plantas más delgadas y pesaron menos), aunque no presentaron cambios en el crecimiento en altura. La expresión de SlXTH1 y la actividad XET total fue particularmente importante en los tejidos vasculares. Nuestros resultados sugieren que el gen SlXTH1 podría estar implicado en la diferenciación de los haces vasculares. 4. Los frutos de las plantas que sobre-expresaron el gen SlXTH1 cuentan con una mayor masa molecular del xiloglucano y fueron mas duros, presumiblemente por los cambios encontrados en la estructura del xiloglucano provocados por la actividad XET, lo que sugiere que esta enzima es clave para el mantenimiento de la estructura del xiloglucano y de la pared celular. 5. La respuesta a los estreses abióticos ensayados de las plantas que tenían reprimida la expresión del gen SlXTH1, fue una disminución del crecimiento, sugiriendo la implicación del gen SlXTH1 en las modificaciones del xiloglucano, ocasionando diferencias en la estructura de la pared celular que alterarán la respuesta a estos estreses. 6. En la respuesta de los frutos a los estreses bióticos, la expresión génica de todas las SlXTHs estudiadas y de la actividad XET se redujo. Por lo tanto, si la actividad XET es inhibida durante la infección, el papel mantenedor de la estructura del xiloglucano se anula, ocasionando el ablandamiento del fruto y el avance de la infección fúngica. En este caso, se sugiere la posibilidad de que se produjera una regulación de origen fúngico a nivel molecular de los genes SlXTHs. 7. El etileno exógeno aplicado a frutos, indujo un aumento de la expresión de todas las SlXTHs estudiadas, mientras que la actividad XET total disminuyó. Por lo tanto, la disminución de la expresión génica de las SlXTHs, durante los procesos de la maduración e infección fúngica, no parece ser debida al aumento del etileno endógeno. De modo que, no podemos explicar qué implicaciones fisiológicas tiene el aumento de la expresión de los genes SlXTHs provocado por el etileno exógeno, lo que hace necesario seguir investigando los procesos de regulación de esta enzima. __________________________________________________________________________________________________The main object of this Thesis doctoral work was based in the physiological, biochemical and molecular characterization of the xyloglucan endotransglucosilase / hidrolase enzime. To achieve this, we have worked with tomato plants (Solanum lycopersicum L.) as a model system of Money Maker and Canario variety and transgenics plants, which have over-expression and repression level of expression of SlXTH1. On the one hand, our purpose was the study of the participation of XET in the plants growing, elongation and cellular expansion. After that, the second partial objective, was based on clarifying the implication of XET in fruits growing and softening. Moreover, it was to assess the paper of enzimes implicated in the cell wall metabolism of fruits during infection by fungus pathogen and in particulary, the implication of XET. Finally, we had studied the possible regulation of XTHs by ethylene. The most relevant conclusions were the following items: 1. This is the first time that transgenics plants which have increased the levels of expression of XTH gene and show an increase in the enzimatic XET activity have been described. 2. In seedling, the sobre-expression of SlXTH1 gene, mainly increases the XET soluble activity, which implies irreversible and permanent changes in the links of xyloglucan with the rest of components, facts that were responsible of an increase of extensibility in epycotils. These results suggest that XET is implicated in growing. 3. In plants that have a decrease on the expression of SlXTH1 gene, changes in the fenotype it were found (plant slimmer and lighter), although these plants reached the same height. These results suggest that SlXTH1 gene would be implicated in vascular veins differentiation. 4. The fruits of plants that have sobre-expression SlXTH1 gene were harder and had higher molecular mass that wild fruits, due to changes in xyloglucan structure caused by XET activity. This suggests that XET is implicated in supporting the structure of cell wall. 5. The genetic expression of SlXTHs and activity XET decreased in fruits which suffer from biotic stress (fungus infection). In this case, we suggest the possibility of a fungus molecular regulation in SlXTHs genes

The role of the secondary cell wall in plant resistance to pathogens

Plant resistance to pathogens relies on a complex network of constitutive and inducible defensive barriers. The plant cell wall is one of the barriers that pathogens need to overcome to successfully colonize plant tissues. The traditional view of the plant cell wall as a passive barrier has evolved to a concept that considers the wall as a dynamic structure that regulates both constitutive and inducible defense mechanisms, and as a source of signaling molecules that trigger immune responses. The secondary cell walls of plants also represent a carbon-neutral feedstock (lignocellulosic biomass) for the production of biofuels and biomaterials. Therefore, engineering plants with improved secondary cell wall characteristics is an interesting strategy to ease the processing of lignocellulosic biomass in the biorefinery. However, modification of the integrity of the cell wall by impairment of proteins required for its biosynthesis or remodeling may impact the plants resistance to pathogens. This review summarizes our understanding of the role of the plant cell wall in pathogen resistance with a focus on the contribution of lignin to this biological process

Disruption of abscisic acid signaling constitutively activates Arabidopsis resistance to the necrotrophic fungus Plectosphaerella cucumerina.

Plant resistance to necrotrophic fungi is regulated by a complex set of signaling pathways that includes those mediated by the hormones salicylic acid (SA), ethylene (ET), jasmonic acid (JA), and abscisic acid (ABA). The role of ABA in plant resistance remains controversial, as positive and negative regulatory functions have been described depending on the plant-pathogen interaction analyzed. Here, we show that ABA signaling negatively regulates Arabidopsis (Arabidopsis thaliana) resistance to the necrotrophic fungus Plectosphaerella cucumerina. Arabidopsis plants impaired in ABA biosynthesis, such as the aba1-6 mutant, or in ABA signaling, like the quadruple pyr/pyl mutant (pyr1pyl1pyl2pyl4), were more resistant to P. cucumerina than wild-type plants. In contrast, the hab1-1abi1-2abi2-2 mutant impaired in three phosphatases that negatively regulate ABA signaling displayed an enhanced susceptibility phenotype to this fungus. Comparative transcriptomic analyses of aba1-6 and wild-type plants revealed that the ABA pathway negatively regulates defense genes, many of which are controlled by the SA, JA, or ET pathway. In line with these data, we found that aba1-6 resistance to P. cucumerina was partially compromised when the SA, JA, or ET pathway was disrupted in this mutant. Additionally, in the aba1-6 plants, some genes encoding cell wall-related proteins were misregulated. Fourier transform infrared spectroscopy and biochemical analyses of cell walls from aba1-6 and wild-type plants revealed significant differences in their Fourier transform infrared spectratypes and uronic acid and cellulose contents. All these data suggest that ABA signaling has a complex function in Arabidopsis basal resistance, negatively regulating SA/JA/ET-mediated resistance to necrotrophic fungi

Signwalling: signals derived from arabiopsis cell wall activate specific resistance to pathogens

The cell wall is a dynamic structure that regulates both constitutive and inducible plant defence responses. Different molecules o DAMPs (damage-associated molecular patterns) can be released from plant cell walls upon pathogen infection or wounding and can trigger immune responses. To further characterize the function of cell wall on the regulation of these immune responses, we have performed a biased resistance screening of putative/well-characterized primary/secondary Arabidopsis thaliana cell wall mutants (cwm). In this screening we have identified more than 20 cwm mutants with altered susceptibility/resistance to at least one of the following pathogens: the necrotrophic fungi Plectosphaerella cucumerina, the vascular bacterium Ralstonia solanacearum, the biotrophic oomycete Hyaloperonospora arabidopsidis and the powdery mildew fungus Erisyphe cruciferarum. We found that cell wall extracts from some of these cwm plants contain novel DAMPs that activate immune responses and conferred enhanced resistance to particular pathogens when they were applied to wild-type plants. Using glycomic profiling we have performed an initial characterization of the active carbohydrate structures present in these cwm wall fractions, and we have determined the signalling pathways regulated by thesse fractions. . The data generated with this collection of wall mutants support the existence of specific correlations between cell wall structure/composition, resistance to particular type of pathogens and plant fitness. Remarkably, we have identified specific cwm mutations that uncoupled resistance to pathogens from plant trade-offs, further indicating the plasticity of wall structures in the regulation of plant immune responses

YODA MAPK kinase kinase regulates a novel immunity pathway conferring broad-spectrum resistance to pathogens

Plant mitogen-activated protein kinase (MAPK) casca des transduce environmental molecular signals and developmental cues into cellular responses. Among these signals are the pathogen-associated molecular patterns (PAMPs) that upon recognition by plant pattern recognition receptors (PRR), including Receptor-Like Kinases (RLKs), activate MAPK cascades that regulate PAMP-triggered immunity responses (PTI)