Involvement of a rice mutation in storage protein biogenesis in endosperm and its genomic location.

MAIN CONCLUSION: A mutation was first found to cause the great generation of glutelin precursors (proglutelins) in rice (Oryza sativa L.) endosperm, and thus referred to as GPGG1. The GPGG1 was involved in synthesis and compartmentation of storage proteins. The PPR-like gene in GPGG1-mapped region was determined as its candidate gene. In the wild type rice, glutelins and prolamins are synthesized on respective subdomains of rough endoplasmic reticulum (ER) and intracellularly compartmentalized into different storage protein bodies. In this study, a storage protein mutant was obtained and characterized by the great generation of proglutelins combining with the lacking of 13 kD prolamins. A dominant genic-mutation, referred to as GPGG1, was clarified to result in the proteinous alteration. Novel saccular composite-ER was shown to act in the synthesis of proglutelins and 14 kD prolamins in the mutant. Additionally, a series of organelles including newly occurring several compartments were shown to function in the transfer, trans-plasmalemmal transport, delivery, deposition and degradation of storage proteins in the mutant. The GPGG1 gene was mapped to a 67.256 kb region of chromosome 12, the pentatricopeptide repeat (PPR)-like gene in this region was detected to contain mutational sites.

Imaging the ER and Endomembrane System in Cereal Endosperm.

The cereal endosperm is a complex structure comprising distinct cell types, characterized by specialized organelles for the accumulation of storage proteins. Protein trafficking in these cells is complicated by the presence of several different storage organelles including protein bodies (PBs) derived from the endoplasmic reticulum (ER) and dynamic protein storage vacuoles (PSVs). In addition, trafficking may follow a number of different routes depending on developmental stage, showing that the endomembrane system is capable of massive reorganization. Thus, developmental sequences involve progressive changes of the endomembrane system of endosperm tissue and are characterized by a high structural plasticity and endosomal activity.Given the technical dexterity required to access endosperm tissue and study subcellular structures and SSP trafficking in cereal seeds, static images are the state of the art providing a bulk of information concerning the cellular composition of seed tissue. In view of the highly dynamic endomembrane system in cereal endosperm cells, it is reasonable to expect that live cell imaging will help to characterize the spatial and temporal changes of the endomembrane system. The high resolution achieved with electron microscopy perfectly complements the live cell imaging.We therefore established an imaging platform for TEM as well as for live cell imaging. Here, we describe the preparation of different cereal seed tissues for live cell imaging concomitant with immunolocalization studies and ultrastructure.

Xyloglucan side chains enable polysaccharide secretion to the plant cell wall.

Plant cell walls are essential for growth. The cell wall hemicellulose xyloglucan (XyG) is produced in the Golgi apparatus before secretion. Loss of the Arabidopsis galactosyltransferase MURUS3 (MUR3) decreases XyG d-galactose side chains and causes intracellular aggregations and dwarfism. It is unknown how changing XyG synthesis can broadly impact organelle organization and growth. We show that intracellular aggregations are not unique to mur3 and are found in multiple mutant lines with reduced XyG D-galactose side chains. mur3 aggregations disrupt subcellular trafficking and induce formation of intracellular cell-wall-like fragments. Addition of d-galacturonic acid onto XyG can restore growth and prevent mur3 aggregations. These results indicate that the presence, but not the composition, of XyG side chains is essential, likely by ensuring XyG solubility. Our results suggest that XyG polysaccharides are synthesized in a highly substituted form for efficient secretion and then later modified by cell-wall-localized enzymes to fine-tune cell wall properties.

Genetic Screening of Factors in the Plant Protein Secretion.

The endomembrane system in plants is composed of interconnected membrane organelles that contribute to intracellular structure and function. These organelles include the endoplasmic reticulum (ER), Golgi apparatus, vacuole, trans-Golgi network, and prevacuolar compartment or multivesicular body. Through vesicle-mediated transport, secreted proteins are synthesized in the ER and subsequently transported along the secretory pathway to the vacuole or outside of cells to fulfill specialized functions. Genetic screening is a crucial method for studying plant protein secretion. It entails identifying phenotypic differences resulting from genetic mutations, such as ethyl methanesulfonate, T-DNA insertion, and RNAi, to investigate gene function and discover mutants with specific traits or gene functions. Significant progress has been achieved in the study of plant protein secretion through genetic screening. In this protocol, we provide a step-by-step guide to studying the protein secretion pathway using a genetic screen approach. We use the example of the free 1 suppressor of Arabidopsis thaliana and oil body mutants of Marchantia polymorpha. Additionally, we offer an overview of genetic screening and briefly summarize the emerging technologies in the field of protein secretion research.

Transmembrane protein TMEM230, regulator of metalloproteins and motor proteins in gliomas and gliosis.

Glial cells provide physical and chemical support and protection for neurons and for the extracellular compartments of neural tissue through secretion of soluble factors, insoluble scaffolds, and vesicles. Additionally, glial cells have regenerative capacity by remodeling their physical microenvironment and changing physiological properties of diverse cell types in their proximity. Various types of aberrant glial and macrophage cells are associated with human diseases, disorders, and malignancy. We previously demonstrated that transmembrane protein, TMEM230 has tissue revascularization and regenerating capacity by its ability to secrete pro-angiogenic factors and metalloproteinases, inducing endothelial cell sprouting and channel formation. In healthy normal neural tissue, TMEM230 is predominantly expressed in glial and marcophate cells, suggesting a prominent role in neural tissue homeostasis. TMEM230 regulation of the endomembrane system was supported by co-expression with RNASET2 (lysosome, mitochondria, and vesicles) and STEAP family members (Golgi complex). Intracellular trafficking and extracellular secretion of glial cellular components are associated with endocytosis, exocytosis and phagocytosis mediated by motor proteins. Trafficked components include metalloproteins, metalloproteinases, glycans, and glycoconjugate processing and digesting enzymes that function in phagosomes and vesicles to regulate normal neural tissue microenvironment, homeostasis, stress response, and repair following neural tissue injury or degeneration. Aberrantly high sustained levels TMEM230 promotes metalloprotein expression, trafficking and secretion which contribute to tumor associated infiltration and hypervascularization of high tumor grade gliomas. Following injury of the central nervous or peripheral systems, transcient regulated upregulation of TMEM230 promotes tissue wound healing, remodeling and revascularization by activating glial and macrophage generated microchannels/microtubules (referred to as vascular mimicry) and blood vessel sprouting and branching. Our results support that TMEM230 may act as a master regulator of motor protein mediated trafficking and compartmentalization of a large class of metalloproteins in gliomas and gliosis.