Cardosin B PSI Transiently Expressed in Arabidopsis Lines with Alterations of ER Structure Is Secreted to the Extracellular Space in Cotyledon Cells

As the main transport route in eukaryotic cells the secretory pathway involves several membranous compartments including the Endoplasmic Reticulum (ER), the Golgi apparatus and Vacuoles. The ER is considered the hub of the secretory pathway, as newly synthesized proteins are distributed to other organelles from the ER. Another vital transport route is the endocytic pathway, which occurs at the plasma membrane and allows the recycling of membranes and membrane proteins. It has been suggested that secretory and endocytic routes may share intermediate compartments ER-related. Therefore, our main goal was to identify any cell compartment common to both routes, combining mutants with altered ER structure with the expression of known vacuolar markers. The Arabidopsis thaliana mutants nai GFP-h and leb-2 GFP-h, presenting alterations of the ER morphology, were selected from the NASC database. Leaf cells ultrastructure was characterized through Transmission Electron Microscopy revealing huge well-defined starch granules in chloroplasts of both seedling and mature leaves of leb-2. The mutated lines were transiently transformed with PSI-B fused with mCherry to analyze the vacuolar routes. Observation by Confocal Microscopy showed that PSI-B is secreted in nai GFP-h and, to a lesser extent, in leb-2 GFP-h. In a secretion assay the PSI-B was found in the extracellular protein content. Given that PSI-B mediates trafficking to the vacuole, it is intriguing to find it extracellularly in these mutant lines. Further observations are required to understand the effect of these mutations in protein vacuolar transport and implications on secretory and endocytic pathways.