Gibberellic Acid-Induced Aleurone Layers Responding to Heat Shock or Tunicamycin Provide Insight into the N-Glycoproteome,Protein Secretion,and Endoplasmic Reticulum Stress

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.Plant proteins that are secreted to the apoplast have important functions in signaling, defense, and cell regulation. The classical protein secretory pathway is less characterized in plants than in mammals or yeast but is of growing interest due to the potential of plant systems as hosts for the production of recombinant proteins (Erlendsson et al., 2010; De Wilde et al., 2013). Plant secretomics, therefore, is a rapidly expanding area applied to gain further insight into these processes (Agrawal et al., 2010; Alexandersson et al., 2013). Many secretory proteins contain putative N-glycosylation sites, and the identification and characterization of these sites is an important element in secretomics analysis. However, to date, only a few plant glycoproteomes have been described (Fitchette et al., 2007; Minic et al., 2007; Palmisano et al., 2010; Melo-Braga et al., 2012; Zhang et al., 2012; Thannhauser et al., 2013).The cereal aleurone layer is of major importance due to its central role in grain germination. Previous proteomic studies have been reported for aleurone layers dissected from mature (Finnie and Svensson, 2003) and germinating (Bønsager et al., 2007) barley (Hordeum vulgare) or developing (Tasleem-Tahir et al., 2011) and mature (Laubin et al., 2008; Jerkovic et al., 2010; Meziani et al., 2012) wheat (Triticum aestivum) grains. The in vitro culture of isolated aleurone layers was developed by Chrispeels and Varner (1967). Since then, this system has become an excellent tool for the study of germination signaling in response to phytohormones (Bush et al., 1986; Jones and Jacobsen, 1991; Bethke et al., 1997; Ishibashi et al., 2012). More recently, it has been adopted as a unique system for the analysis of plant secretory proteins (Hägglund et al., 2010; Finnie et al., 2011). The addition of GA₃ to the isolated aleurone layers induces the synthesis and secretion of hydrolytic enzymes. In the in vitro system, these accumulate in the incubation buffer, facilitating their identification and characterization using proteomics techniques. Thus, numerous secreted proteins with roles in the hydrolysis of starch, cell wall polysaccharides, and proteins could be identified (Finnie et al., 2011). Furthermore, several of the proteins were also detected in intracellular extracts from the same aleurone layers, presumably prior to their release. Many of the proteins appeared in multiple forms on two-dimensional (2D) gels, and often with higher M_r than expected, suggesting the presence of posttranslational modifications (PTMs; Finnie et al., 2011). Bak-Jensen et al. (2007) and Finnie et al. (2011) observed a highly complex pattern of α-amylase-containing spots on 2D gels, which originated from only two α-AMYLASE2 (AMY2) and two AMY1 gene products from a total of 10 genes, probably reflecting multiple forms due to PTMs.In eukaryotic cells, proteins synthesized in the endoplasmic reticulum (ER) must be correctly folded and assembled before continuing in the secretory pathway. Perturbations of redox state, calcium regulation, Glc deprivation, and viral infection can lead to ER stress, triggered by the accumulation of unfolded and misfolded proteins in the ER lumen. This provokes a triple response from the cell, consisting of an up-regulation of chaperones and vesicle trafficking, a down-regulation of genes encoding secretory proteins, and an up-regulation of proteins involved in endoplasmic reticulum-associated protein degradation (ERAD). In plants, the molecular mechanisms underlying ER stress in plants have yet to be fully resolved (Martínez and Chrispeels, 2003; Nagashima et al., 2011; Moreno et al., 2012).Tunicamycin (TN), an inhibitor of GlcNAc phosphotransferase, which catalyzes the first step in glycoprotein synthesis, has been used to induce ER stress by causing the accumulation of unfolded proteins in the ER lumen (Noh et al., 2003; Kamauchi et al., 2005; Reis et al., 2011). If unfolded proteins are not removed, the prolonged stress will induce programmed cell death. Links between ER stress and apoptosis have been reported in response to TN treatment in mammalian cells, whereas in plants, this correlation has been suggested, but the pathways of signal transduction remain unknown (Kamauchi et al., 2005; Reis et al., 2011).In all plant tissues, heat shock (HS) induces the synthesis of a variety of heat shock proteins (HSPs), which are responsible for protein refolding under stress conditions (Craig et al., 1994) and translocation and degradation in a broad array of normal cellular processes (Bond and Schlesinger, 1986; Spiess et al., 1999). In the barley aleurone layer, HS selectively suppresses the synthesis of secretory proteins, including α-amylase, due to the selective destabilization of secretory protein mRNA (Belanger et al., 1986; Brodl and Ho, 1991). However, an acclimation effect has been described in aleurone cells after prolonged incubation at warm temperatures, resulting in a resumption of the protein secretory machinery (Shaw and Brodl, 2003). The connection between heat stress response and ER stress has been well established in mammals and yeast, but scarce information is available in plants (Denecke et al., 1995).Over the last years, the dual role of reactive oxygen species (ROS) has been established in plants: at higher concentrations, ROS act as toxic molecules damaging cellular macromolecules, eventually causing cell death, but at lower concentrations, ROS seem to be necessary for seed germination and seedling growth by controlling the cellular redox status, regulating growth and protecting against pathogens (Bailly, 2004; Bailly et al., 2008). In the barley aleurone layer, GA₃ perceived at the plasma membrane induces ROS generation as a by-product from intense lipid metabolism, and the redox regulation of the GA₃-induced response has been proposed (Maya-Ampudia and Bernal-Lugo, 2006). This suggests that during the secretory function of the tissue, moderate levels of ROS may be acting as cellular messengers. In aleurone cells, ROS, especially hydrogen peroxide (H₂O₂), are involved in the process of programmed cell death, but the molecular mechanisms remain unclear (Bethke and Jones, 2001; Ishibashi et al., 2012).Until now, none of the protein components of the ER stress pathways have been identified in barley; also, little is known about the glycosylation of barley proteins. In this work, numerous N-glycoslation sites are identified, and the effect of perturbing N-glycosylation and the secretory pathway by TN and HS treatments is analyzed in GA₃-induced barley aleurone layers.