Functional analysis of a phloem cysteine protease in arabidopsis thaliana
- Autores: Eugenia Pitsili
- Directores de la Tesis: Núria Sánchez Coll (dir. tes.), Marc Valls Matheu (tut. tes.)
- Lectura: En la Universitat de Barcelona ( España ) en 2021
- Idioma: inglés
- Tribunal Calificador de la Tesis: Miguel Ángel Moreno Risueño (presid.), Salomé Prat (secret.), Hannele Tuominen (voc.)
- Programa de doctorado: Programa de Doctorado en Biotecnología por la Universidad de Barcelona
- Materias:
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- Resumen
Metacaspases are a family of cysteine proteases found in lower eukaryotes and plants. They are considered to be distant relatives of caspases, present only in animals. The role of metacaspases is quite diverse, from developmental processes involved in tissue formation, to responses against pathogens and other environmental stresses. For some members of the family there is ongoing research regarding their function and possible substrates and for some others they have not been characterized.
In this study we attempted to functionally analyse Arabidopsis thaliana metacaspase 3 (MC3), an uncharacterized metacaspase which was found to be expressed in the vascular tissue. We considered that the best way to address this challenge was to use a reverse genetics approach in combination with proteomic analysis. In Chapter 2, we specifically identified the exact localization of the transcript and the protein in planta, we generated mutant plants using the CRISPR/Cas9 technology and we analysed their growth in basal conditions. Moreover, we performed a whole proteome analysis for different tissues and we attempted to identify candidate substrates by N-termini analysis. In Chapter 3, we delved into multiple stresses testing how plants with over-accumulation or lack of MC3 responded to stress conditions, since multiple proteins related to stress responses came up from the proteomic study.
In Chapter 2 we generated reporter lines to specify the exact localization of the gene expression. MC3 was found specifically expressed in the phloem vascular tissue. Furthermore, translational fusion to fluorescent proteins verified the same pattern for protein localization. Using multiple mutant lines, we performed an analysis of the phenotypes caused from the absence, malfunction or overexpression of the putative protease in the development of the plant. Overall growth and formation of the vascular tissue in particular, were not affected when plants were grown upon standard growth conditions. From previous studies, MC1 reporter lines exhibited expression in the stele in almost every developmental stage and MC4 has a high and ubiquitous expression in most of the tissues.
Double mutants were generated in order to exclude the possibility of functional redundancy and we showed that under normal conditions the overall growth of these plant remained similar to wild-type plants. Finally, we checked the total proteome of the plants to compare the differentially abundant proteins in different expression backgrounds. Overall, the analysis demonstrated that root tissue of MC3 overexpressor lines showed differential accumulation of stress related proteins, specifically osmotic and hypoxic related.
Metacaspases have been shown to participate amongst others, in the plant responses to biotic and abiotic stresses. In Chapter 3 we analyzed whether MC3 had a role in responses to different environmental stresses. We found that MC3 function is associated with drought stress, since plants overaccumulating MC3 were able to survive more and performed better under low water availability conditions. Moreover, compared to wild-type plants, mc3 mutant appeared less sensitive to ABA, which is one of the hormones orchestrating drought responses. Considering that the vasculature plays a very important role in facilitating ABA signalling, we investigated vascular formation upon osmotic stress in plants with altered MC3 levels. Overexpressor lines showed a faster formation of the vascular tissue under osmotic stress conditions than wild type plants. Finally, we observed that in conditions of low oxygen concentrations, the excessive amount of MC3 can also be beneficial for plant survival. Additional stresses were tested in order to detect if MC3 function was specific of osmotic unbalances. Plants were not affected of the presence or absence of MC3 upon light and temperature altered conditions, neither upon infection with a vascular pathogen. In conclusion, we reported that metacaspase 3 is a phloem-specific metacaspase which contributes to drought tolerance possibly due to enhanced metaphloem vascular differentiation upon osmotic stress conditions.
