PER ASPERA - Plant Endoplasmic Reticulum Architecture and Seed Productivity

The plant endoplasmic reticulum (ER) is the cellular organelle that regulates the flux of proteins and lipids into the secretory pathway, and is responsible for storing large amount of proteins for human and animal nutrition. The ER has a unique and dynamic architecture, which changes to allow for different biosynthetic functions. Building on our collaborative work over the last 5 years, our team combines expertise in plant molecular and cell biology, biochemistry, microscopy and cereal genetics to bring understanding of ER structure/function relationships to the next level. We aim at understanding the key molecular determinants of ER shape by studying their function, regulation and interactions. We also propose to investigate interorganellar cooperation by analysing putative contact points betweent ER and plasma membrane and ER and protein storage vacuoles. We will manipulate the key ER morphogens and assess how changes in ER shape affect protein and lipid biosynthesis and storage. This work will be perfomed in model plants and, importantly, in seeds of cereals (barley, weat and maize) in order to test directly the ER structure/function relationships in these crop models. The objectives of the project are: 1. To unravel the machinery involved in creating and maintaining a plant ER network through the functional study of known ER shaping proteins and their interactors 2. To analyse plant endoplasmic reticulum structure in depth from macromolecular organisation to 3- D architecture in a range of cell types 3. To establish the mechanisms of ER interactions/contacts with tonoplast and plasma membrane 4. To manipulate the biosynthetic capacity of the ER through manipulation of form 5. To establish barley and wheat as systems to study ER productivity in cereals

The plant endoplasmic reticulum (ER) is the cellular organelle responsible for the synthesis of the majority of seed proteins key to human and animal nutrition. The ER is the gateway of the secretory pathway, which synthesises and exports most storage proteins. It can also act as a protein storing compartment in its own right. Our project aimed at understanding how the unique morphology of the plant endoplasmic reticulum (ER) is formed and maintained, and how ER shape affects its biosynthetic function. We also aimed to extend the fundamental discoveries made in the model plant Arabidopsis into cereal models, barley and maize. We have made very significant progress: we understood the mechanism by which a class of proteins named reticulons shape the ER membrane into tubules, providing the first in vivo evidence for this mechanism. We also identified some reticulons isoforms which have a different function. We studied novel ER morphogenic proteins that maintain the integrity of the ER network. In particular, we identified the plant homologues of a protein called Lunapark, which oversees ER tubular junctions. In the Austrian part of the project we gained new insights into the morphological changes of the endomembrane system during the development of cereal endosperm, and we advanced our understanding of how the ER itself can store proteins in barley and maize seeds. We utilized a model system to study ER- derived protein bodies by inducing their formation in tobacco leaves. Using this system we could also prove the applied relevance of protein bodies for the bio-encapsulation of vaccine- candidates. We were able to show that a model vaccine encapsulated within protein bodies resulted in a superior immune response compared to the soluble antigen. We also initiated work to test how ER morphology can affect the process of ER protein body formation. To this end we studied protein body formation upon overexpression of reticulons and we could show an effect on the size and number of storage organelles. Our project also produced some significant technical advances: the consortium developed high-resolution in vivo confocal microscopy protocols to visualise ER morphology changes, and new computational tools to describe and quantify the morphology of the ER, allowing us to measure the effect of modulating ER morphogen levels on the overall architecture of this organelle. We also used serial blockface electron microscopy to visualise the endomembrane system across whole cellular volumes and to monitor developmental changes in seeds during protein body biogenesis.