Plasma membrane repair mechanisms in plants 

 Abiotic stress episodes such as salinity, drought, and non-optimal temperatures constitute the principal cause of crop yield loss worldwide. To cope with this major constraint to agricultural production, the development of abiotic stress tolerant crops ensuring productivity under sub-optimal conditions has become of paramount importance for plant scientists.

Plants undergo continuous exposure to multiple stresses that singularly or in combination induce cellular damage. To minimize damage, plants recognize environmental cues and trigger physiological, and biochemical reactions aimed to: 1) activate short-term repair mechanisms to avoid cell death due to plasma membrane (PM) disruptions, 2) Induce long-term physiological adaptations to increase the cellular resistance toward stresses, i.e. acclimation. Although classical genetics and systems biology approaches have identified multiple pathways involved in stress acclimation, little is known about the subcellular events and components required to activating cellular repair after PM injury. In this context, our identification of the founding member of the Arabidopsis synaptotagmins gene family (SYT1) is an important advance. The syt mutant is defective in plasma membrane repair under various stress conditions, indicating that SYT1 is a key component in PM repair during stress.  What is not known is how SYT1 participates in the complex mechanism of PM repair, requiring the participation of Ca2+, phospholipids, and multiprotein complexes.

Thus, by investigating the function of the SYT family in abiotic stress tolerance and by identifying potential SYTs interacting partners, we will contribute to a better understanding of how plants modulate the PM repair processes. To achieve this long-term goal, we will use different experimental approaches described below combining tools from the genetics, physiology, molecular biology, bioinformatics, and cell biology fields on the model plant Arabidopsis thaliana.

Role of membrane contact sites (MCS) in abiotic stress tolerance. Bioinformatic studies have shown that plant SYTs harbors a membrane-binding domain found exclusively in proteins localized in MCSs. Our SYT1 immuno- and co-localization studies, suggest that different SYTs might act as a multiprotein complex in MCSs between the PM and the cortical endoplasmic reticulum (cER). We intend to use TEM microscopy and 3D-modeling of the PM-cER contact sites to identify structural features linked to the increased abiotic stress sensitivity on syt mutants.

Analysis of the SYT1 role in non-vesicular phospholipid transfer.  SYT1 is closely associated to the PM and binds negatively charged artificial liposomes in a Ca+2 -dependent mode, however the lipid species bound in vivo has not been characterized. By using lipid-protein overlay assays at different Ca+2 concentrations we will identify specific lipids involved in the interactions with SYT1. Next, we will develop fluorescent markers against those lipids to study putative changes on their distribution and dynamics in syt mutants background.

Identification of SYTs binding partners. In animals, a number of SYTs protein interactors bridging apposed membranes have been identified. Since no SYTs interactors has been identified in plants, we propose to use techniques based on split-GFP markers (BIF/FRET), immunoprecipitation and proteomic analysis using both a transgenic SYT1::SYT1-GFP line and specific anti-SYT antibodies, and CHIP protein assays to identify putative SYTs binding partners.

Suppressor and Targeted screen for additional PM repair components

The Arabidopsis SYTs family is composed by five members. Despite its importance for PM repair after injury, the quintuple SYTs mutant is viable and only displays visible phenotypes under stress conditions. We hypothesize that additional protein components might be involved in PM integrity maintenance on standard conditions. To identify those components we will perform a targeted screen of known mutants involved in related processes such as endocytosis/exocytosis, cytoskeleton organization, phospholipid transport, Ca+2 transport or cell wall biosynthesis using an innovative viability assay that we have developed. We will also perform a non-targeted forward genetic screen looking for suppressors of the syt1 phenotypes under combined NaCl / low Ca+2 stress conditions.