ESCRTing during barley endosperm development

The endosomal sorting complex required for transport (ESCRT)-0, I, II and III promotes ubiquitin-regulated endocytosis where cargo proteins are sorted into multi vesicular bodies (MVB), finally fusing with the vacuole and releasing their content into its lumen. ESCRT proteins are highly conserved and have been shown to carry out a wide range of functions in plant cells: they are required for cytokinesis, development, vacuolar organization and participate in the ER-Golgi mediated protein sorting machinery. In particular ESCRT-III mediates the biogenesis of multivesicular bodies (MVBs) by exerting membrane bending, scission and fusion, and functions in controlling autophagosome fusions, vacuolar integrity and peroxisomal invaginations. The endomembrane system of endosperm tissue is characterized by a high structural plasticity and endosomal activity. Protein trafficking in these cells is complicated by the presence of several different storage organelles including dynamic protein storage vacuoles (PSVs) and protein bodies (PBs) derived from the endoplasmic reticulum (ER). In addition, trafficking may follow a number of different routes, depending on cell type, developmental stage and environment, showing that the endomembrane system is capable of massive reorganization. In view of the highly dynamic endomembrane system in cereal endosperm cells it is reasonable to expect that ESCRT-III participates in cellular processes in this tissue. First bioinformatics results underline this assumption as there are orthologs of the ESCRT-III proteins present in cereals. In addition, it has been shown that SAL1 (ESCRT-III associated complex protein) is indispensable for cellular processes in maize aleurone, indicating a fuctional significance of ESCRT-III in endosperm tissue. This project therefore aims to further our knowledge about the functional organization of ESCRT-III in barley endosperm tissue and its protein sorting role in seed storage protein trafficking in barley endosperm by cell biological, molecular biological, bioinformatic and biochemical studies. As trafficking pathways of proteins and their final storage in vacuoles are still a matter of debate in cereal endosperm, the results of this project will shed light on how ESCRT-III will sort seed storage proteins in barley endosperm. Therefore, the focus of this work is to characterize the spatial distribution of ESCRT-III within the tissue and at the subcellular level and to understand how ESCRT-III will contribute to the sorting of SSPs in barley endosperm. To this end, I will study the co-localization of ESCRT-III with endomembrane markers, identifying possible interactions with parts of the cereal endomembrane system. In addition, I will analyze the barley SSP hordein in ESCRT-III knock down barley seeds. IPs of HosESCRT-III proteins will identify new interaction proteins.

The endosomal sorting complex required for transport (ESCRT)-0, I, II and III functions as protein sorting machinery and is responsible to deliver proteins to their final destinations in cells. ESCRT-III is highly conserved and is responsible for membrane deformation. In detail, ESCRT-III mediates the biogenesis of multivesicular bodies (MVBs) where the sorted proteins are accumulated before they enter the next step of the pathway within the endomembrane system. The protein trafficking in the cereal endosperm tissue is complicated by the presence of several different storage organelles including dynamic protein storage vacuoles (PSVs) and protein bodies (PBs) that accumulate several different seed storage proteins (SSP). The focus of this work was to characterize by cell biological, molecular biological, bioinformatic and biochemical studies the spatial distribution of ESCRT-III within the tissue and at the subcellular level. The aim was to understand how ESCRT-III will contribute to the sorting of seed storage proteins in barley endosperm. The results of this project show that the localization and the putative function of ESCRT-III in barley endosperm depends on the developmental stage and on the cell layer of the endosperm tissue, respectively. There is the indication, that barley ESCRT-III is possibly involved in functions that are barley endosperm specific. This outcome highlights the importance to be more specific. It is indispensable to study the ESCRT-III in various plants and in different cell-layers in the specific tissue. Bioinformatic assays and molecular biology studies show the existence of all ESCRT-III proteins in barley seeds. Transgenic, viable plants have been generated to enable the live cell imaging of several ESCRT-III proteins in combination with diverse marker proteins of the protein trafficking pathway. Confocal imaging of ESCRT-III (HvVPS60, HvSNF7, HvVPS24) proteins show that the localization differs of these proteins between the diverse cell layers of barley endosperm. Additionally, the localization of HvVPS24 changes during seed development. The function of HvVPS60 is also depending on the celllayer.Biochemical experiments revealed a seed storage protein as putative interaction partner of HvVPS24. This is of special interest, as this seed storage protein is only present in barley/wheat/oat seeds. With the interaction studies, new interactions partners of HvVPS24 were identified, however, we could not identify additional barley ESCRT-III subunits so far.