RT Journal Article
SR Electronic
T1 Cytoskeleton members, MVBs and the ESCRT-III HvSNF7s are putative key players for protein sorting into protein bodies during barley endosperm development
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 595108
DO 10.1101/595108
A1 Valentin Roustan
A1 Julia Hilscher
A1 Marieluise Weidinger
A1 Siegfried Reipert
A1 Azita Shabrangy
A1 Claudia Gebert
A1 Bianca Dietrich
A1 Georgi Dermendjiev
A1 Pierre-Jean Roustan
A1 Eva Stoger
A1 Verena Ibl
YR 2019
UL http://biorxiv.org/content/early/2019/03/31/595108.abstract
AB Cereal endosperm is a short-lived tissue adapted for nutrient storage, containing specialized organelles, such as protein bodies (PBs) and protein storage vacuoles (PSVs), for the accumulation of storage proteins. PBs can be used as efficient biotechnological systems to produce high yields of stable recombinant proteins. During development, protein trafficking and storage require an extensive reorganization of the endomembrane system. Consequently, endomembrane-modifying proteins will influence the final grain quality, yield and recombinant protein production. Barley, a cereal crop of worldwide importance for the brewing industry, animal feed and to a lesser extent, human nutrition, has been identified as promising candidate for recombinant protein production. However, little is known about the molecular mechanism underlying endomembrane system remodeling during barley grain development. By using in vivo label-free quantitative proteomics profiling, we quantified 1,822 proteins across developing barley grains. Based on proteome annotation and a homology search, 95 proteins associated with the endomembrane system were identified, and 83 of these exhibited significant changes in abundance during grain development. Clustering analysis allowed characterization of three different development stages; notably, integration of proteomics data with in situ subcellular microscopic analyses showed a high abundance of cytoskeleton proteins associated with acidified protein bodies at the early development stages. Endosomal sorting complex required for transport (ESCRT)-related proteins and their transcripts are most abundant at early and mid-development. Specifically, multivesicular bodies (MVBs), and the ESCRT-III HvSNF7 proteins are associated with protein bodies (PBs) during barley endosperm development. Taken together, our proteomics results specifically identified members of the cytoskeleton, MVBs, and ESCRT as putative key players for protein sorting into PBs during barley endosperm development. These results present a comprehensive overview of proteins involved in the rearrangement of the endomembrane system during barley early grain development and will provide the basis for future work on engineering the endomembrane system to optimize nutrient content and to produce high yields of recombinant proteins.AMSH1associated molecule with the SH3 domain of Stam1A. thalianaArabidopsis thalianaARFADP ribosylation factorBiFCBimolecular Fluorescence ComplementationDAPdays after pollinationDVdense vesicleECelectrical conductivityERendoplasmic reticulumERO1ER oxidoreductin 1ESCRTendosomal sorting complex required for transportFBPAfructose-bisphosphate aldolaseFDRfalse discovery rateGPGolden PromiseGPCgrain protein contentHSP70heat shock protein 70HvHordeum vulgaremEosFPmonomeric Eos fluorescent proteinILVintraluminal vesicleLC-MS/MSliquid chromatography-mass spectrometryMSmass spectrometryMVBmultivesicular bodyPACprecursor-accumulating vesiclePBprotein bodyPCAprincipal-component-analysisPCDprogrammed cell deathPDIprotein disulfide isomerasePMplasma membranePSVprotein storage vacuoleRT-qPCRreal-time quantitative PCRSAL1supernumerary aleurone layer 1SAMS-adenosyl-L-methionineSNF7sucrose nonfermenting protein 7SSPseed storage proteinSTRINGsearch tool for the retrieval of interacting genes/proteinsTEMtransmission electron microscopyTGNtrans-Golgi networkTIP3tonoplast intrinsic protein 3VPEvacuolar processing enzymeVPSvacuolar protein sortingVSR1vacuolar-sorting receptor 1WMwortmanninY2Hyeast two-hybrid