Functional study of vacular sorting receptors in transgenic "Arabidopsis thaliana" plants

Fusion of the Green Fluorescence protein (GFP) to propeptides of different vacuolar proteins like barley aleurain and tobacco chitinase allowed to visualize two different vacuolar compartments with different sizes in different tissues. These propeptides contain vacuolar sorting determinant (VSD) of two different types: sequence-specific (aleurain) and C-terminal (chitinase). These VSDs are supposed to be recognized by receptors such as VSRs and RMRs. VSRs are supposed to mediate protein sorting to lytic vacuoles, while RMRs are supposed to mediate protein sorting to storage vacuoles. Partial cDNA sequences for these vacuolar sorting receptors were cloned into a geminivirus silencing vector, and introduced by biolistics into transgenic Arabidopsis plants expressing either Aleu-GFP or GFP-chi to visualize effects of gene silencing. The inactivation of the subfamily AtVSR3 in Aleu-GFP transgenic plants caused the absence of the GFP in the large central vacuole in epidermal cells (which are lytic vacuoles) of rosette leaves, while GFP appeared in small compartments which can be ER or Prevacuolar compartments (PVC). Silencing of subfamilies AtVSR 1and 2 did not affect strongly GFP distribution in cells. Seeds from these plants were not able to germinate, and scanning electron micrographs showed that seed coat cells were no more hexagonal and miss their columella compared to Wild type seeds. Unexpectedly, silencing of RMRs in Aleu-GFP plants lead to the secretion of GFP from mesophyll cells. In GFP-chi plants, RMRs silencing also lead to the secretion of the GFP into the extracellular space in mesophyll cells .In these plants, silencing of the VSR subfamilies did not affect the GFP fluorescent in epidermal cell vacuoles. Therefore we confirmed that VSRs and specially the subfamily 3 is the best candidate for sorting of proteins with sequence- specific VSDs in leaves while RMRs seem to be involved in the sorting in both pathways. Also interesting is the used of reverse genetic to study RMRs. This technic was used because of symptoms obtained with germinivirus. Using in situ hybridization, I have detected VSRs receptors in leaves and in roots of Arabiopsis thaliana plants. These results showed that AtVSR 1 and 5 mRNA were the most transcribed in leaves and in root. Finally, it seems that direct interaction between VSRs and RMRs is necessary to sort proteins to lytic vacuoles.

Thèse de doctorat : Université de Neuchâtel, 2006 ; Th. 1963