ESCRT-III Transcriptome and Proteome of Barley Endosperm

Development relies on progressive changes in gene expression as cell clusters, tissues and cell types are determined and differentiate. The cereal endosperm is a complex structure made up of different tissues with distinct cell types. Each cell type has a particular function that is directed by its own unique transcriptome and proteome. The Endosomal sorting complex required for transport (ESCRT) consists of four subunits 0, I, II, and III and is responsible for endocytic recycling of membrane proteins. ESCRT proteins are strongly conserved and are involved in many different cellular processes. It was shown that ESCRT proteins are indispensable for maize endosperm development. Recently we have identified barley ESCRT-III members in the (model) crop Hordeum vulgare (Hv) and show that all known members are expressed in developing barley endosperm. Our initial studies of ESCRT-III in barley endosperm showed that the localization and involvement of an ESCRT-III protein in diverse trafficking pathways is depending on the cell layer. This result is accompanied by the spatio- temporal endomembrane rearrangement in developing barley endosperm, especially between 8 and 12 days after pollination. This project aims to be highly specific by assaying the subcellular ESCRT-III transcriptome and proteome of developing barley endosperm. We will use the Leica Laser microdissection microscope (AS LMD) for spatio-temporal sampling with subsequent gene expression and protein analyses. Thus, we will use the AS LMD for subcellular sampling for analyzing the ESCRT-III proteome by LC-MS. Quantitative proteomic studies will include the analyses of different intracellular trafficking processes related to spatio-temporal regulated HvESCRT-III. Within this project we will perform a multidisciplinary approach for studying the expression profiles in specific cells strengthened by subcellular proteomics: live-cell imaging, RNA and protein studies will generate information on the interacting proteome of ESCRT-III at cellular resolution.