Analysis of the Arabidopsis synaptotagmin gene expression and protein accumulation during stress using luciferase-based markers

The responses of plants to adverse environmental conditions require the adequate sensing of stress signals from the environment and their integration into coordinated cellular responses. These coordinated responses in plants often involve information exchanges between intracellular organelles that rely on vesicular trafficking and/or non-vesicular transport at membrane contact sites. This thesis focuses on the plant endoplasmic reticulum (ER) plasma membrane (PM) contact sites (EPCS), which are evolutionarily conserved nanostructures that facilitate exchanges of information between the cortical ER and the PM. The close association between membranes at EPCS is facilitated by physical tethering often mediated by lipid-transfer proteins. Members of one of these families, known as the plant synaptotagmins, are important elements conferring tolerance to biotic and abiotic stresses in Arabidopsis. Still, little is known about the regulatory mechanisms and environmental triggers controlling the expression and activity of the different synaptotagmins in plants. In this thesis, I use luciferase-based markers to perform systematic analyses of the SYNAPTOTAGMIN1 (SYT1) promoter activity and protein accumulation in Arabidopsis. My results show that SYT1 expression increases in response to salt-induced ionic and osmotic stresses, particularly when the stress generator contains Cl-. Remarkably, the amount of SYT1 protein does not change in response to salt stress indicating that SYT1 transcriptional and translational levels are uncoupled. By measuring the stability of SYT1 in response to NaCl, I determined that salt stress has an increased effect on the stability of the SYT1 protein and that its degradation is partially controlled by the 26S ubiquitin proteasome. I conducted an in silico analyses of gene expression for different members of the Arabidopsis synaptotagmin family and validated the results using SYT promoter - GUS fusions. My results show that while some Arabidopsis SYTs are ubiquitous, some others are expressed in specific tissues, and that their expression is environmentally and developmentally controlled. My results advance our understanding of the expression and regulation of individual SYTs in Arabidopsis and will facilitate the study of their specific contribution/s to the overall stress tolerance in plants.Science, Faculty ofBotany, Department ofGraduat