Abel Rosado Rey

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.

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Plants are sessile and are exposed to environmental changes that can compromise their survival and yield. Salt stress is one of the most common environmental stresses. Arabidopsis SYT1 is a key player in plant response to salt stress mediated by Ca²⁺, and acts as protein bridge, tethering the endoplasmic reticulum to the plasma membrane. These regions of close contact between organelles, called membrane contact sites, are conserved in eukaryotes, and are involved in functions such as Ca²⁺ signaling and lipid homeostasis. However, their relationship with salt stress in plants is unknown. In this project, I aimed to discover new contact site tethers in the model organism Arabidopsis thaliana and shed light on the relation of endoplasmic reticulum-plasma membrane contact sites (EPCS) and salt stress tolerance. I used bioinformatic, phylogenetic and cellular biological approaches and I related an Arabidopsis protein family, the N-terminal transmembrane C2 domain (NTMC2) family, to contact sites. A putative tether called Arabidopsis thaliana Ca²⁺-dependent Lipid Binding (AtCLB) belongs to the NTMC2 family. AtCLB has a subcellular localization pattern of beads and strings, which resembles the pattern found for the plant EPCS tether SYT1. AtCLB pattern becomes more punctate under depletion of cytosolic Ca²⁺, suggesting that intracellular Ca²⁺ is important for AtCLB contact with the plasma membrane. Mutant plants lacking functional AtCLB did not show any visible phenotype in salt stress conditions. Under salt stress treatments, however, the normal subcellular pattern of AtCLB was altered, EPCS formation was increased and the intermembrane distance was decreased. This result suggests a role for AtCLB in salt stress tolerance that might be phenotypically masked by functional redundancy. This subcellular alteration and the reduction of intermembrane distance was mimicked when the amount of phosphatidylinositol-(4,5)-bisphosphate was artificially increased at the plasma membrane. These results point towards a model of EPCS tethering involving cytosolic Ca²⁺, salt stress and negatively charged phosphoinositides. Further study will be required to fully understand EPCS regulation. In summary, these discoveries clarify some mechanistic aspects of EPCS tethering in plants and open a door to the discovery of new contact site protein tethers from the plant protein family NTMC2

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