title: Gibberellic acid-induced aleurone layers responding to heat shock or tunicamycin provide insight into the N-glycoproteome, protein secretion, and endoplasmic reticulum stress.
abstract: The growing relevance of plants for the production of recombinant proteins makes understanding the secretory machinery, including the identification of glycosylation sites in secreted proteins, an important goal of plant proteomics. Barley (Hordeum vulgare) aleurone layers maintained in vitro respond to gibberellic acid by secreting an array of proteins and provide a unique system for the analysis of plant protein secretion. Perturbation of protein secretion in gibberellic acid-induced aleurone layers by two independent mechanisms, heat shock and tunicamycin treatment, demonstrated overlapping effects on both the intracellular and secreted proteomes. Proteins in a total of 22 and 178 two-dimensional gel spots changing in intensity in extracellular and intracellular fractions, respectively, were identified by mass spectrometry. Among these are proteins with key roles in protein processing and secretion, such as calreticulin, protein disulfide isomerase, proteasome subunits, and isopentenyl diphosphate isomerase. Sixteen heat shock proteins in 29 spots showed diverse responses to the treatments, with only a minority increasing in response to heat shock. The majority, all of which were small heat shock proteins, decreased in heat-shocked aleurone layers. Additionally, glycopeptide enrichment and N-glycosylation analysis identified 73 glycosylation sites in 65 aleurone layer proteins, with 53 of the glycoproteins found in extracellular fractions and 36 found in intracellular fractions. This represents major progress in characterization of the barley N-glycoproteome, since only four of these sites were previously described. Overall, these findings considerably advance knowledge of the plant protein secretion system in general and emphasize the versatility of the aleurone layer as a model system for studying plant protein secretion.
authors.affiliation: Agricultural and Environmental Proteomics , Department of Systems Biology, Technical University of Denmark, 2800 Kongens Lyngby, Denmark.
authors.forename: Gregorio
authors.initials: G
authors.lastname: Barba-Espín
authors.forename: Plaipol
authors.initials: P
authors.lastname: Dedvisitsakul
authors.forename: Per
authors.initials: P
authors.lastname: Hägglund
authors.forename: Birte
authors.initials: B
authors.lastname: Svensson
authors.forename: Christine
authors.initials: C
authors.lastname: Finnie
mesh_terms: D016923:Cell Death / Q000187:drug effects; D002457:Cell Extracts; D016000:Cluster Analysis; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D056625:Endosperm / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism; D005453:Fluorescence; D005875:Gibberellins / Q000494:pharmacology*; D006023:Glycoproteins / Q000378:metabolism*; D006031:Glycosylation / Q000187:drug effects; D018869:Heat-Shock Response / Q000187:drug effects*; D001467:Hordeum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism; D015227:Lipid Peroxidation / Q000187:drug effects; D008954:Models, Biological; D010940:Plant Proteins / Q000378:metabolism; D025341:Principal Component Analysis; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics; D013194:Staining and Labeling; D014415:Tunicamycin / Q000494:pharmacology*; D000516:alpha-Amylases / Q000096:biosynthesis
chemical_list: D002457:Cell Extracts; D005875:Gibberellins; D006023:Glycoproteins; D010940:Plant Proteins; D020543:Proteome; D014415:Tunicamycin; D006861:Hydrogen Peroxide; C007842:gibberellic acid; D000516:alpha-Amylases