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 i… Show more
“…These hydrolytic enzymes are secreted to the starchy endosperm to break down starch and protein that accumulated in the starchy endosperm to supply carbon and nitrogen used in germination. Whereas the secretion of α-amylases and cysteine proteases from the aleurone layers increased in response to gibberellic acid treatment for 24 h, the level of the barley TaPDIL-1 ortholog was not affected by gibberellic acid treatment [64], suggesting that the barley TaPDIL-1 ortholog that remains in the aleurone cell of the wheat grain may help fold the newly synthesized hydrolytic enzymes.Figure 8
Distribution of wheat PDI family proteins in the mature aleurone cells and the protein matrix of wheat grains. Cross sections of mature grains were immunostained with sera against TaPDIL1Aα (A, F, K) , TaPDIL2 (B, G, L) , TaPDIL3A (C, H, M) , TaPDIL4D (D, I, N) , or TaPDIL5A (E, J, O) .…”
BackgroundThe major wheat seed proteins are storage proteins that are synthesized in the rough endoplasmic reticulum (ER) of starchy endosperm cells. Many of these proteins have intra- and intermolecular disulfide bonds. In eukaryotes, the formation of most intramolecular disulfide bonds in the ER is thought to be catalyzed by protein disulfide isomerase (PDI) family proteins. The cDNAs that encode eight groups of bread wheat (Triticum aestivum L.) PDI family proteins have been cloned, and their expression levels in developing wheat grains have been determined. The purpose of the present study was to characterize the enzymatic properties of the wheat PDI family proteins and clarify their expression patterns in wheat caryopses.ResultsPDI family cDNAs, which are categorized into group I (TaPDIL1Aα, TaPDIL1Aβ, TaPDIL1Aγ, TaPDIL1Aδ, and TaPDIL1B), group II (TaPDIL2), group III (TaPDIL3A), group IV (TaPDIL4D), and group V (TaPDIL5A), were cloned. The expression levels of recombinant TaPDIL1Aα, TaPDIL1B, TaPDIL2, TaPDIL3A, TaPDIL4D, and TaPDIL5A in Escherichia coli were established from the cloned cDNAs. All recombinant proteins were expressed in soluble forms and purified. Aside from TaPDIL3A, the recombinant proteins exhibited oxidative refolding activity on reduced and denatured ribonuclease A. Five groups of PDI family proteins were distributed throughout wheat caryopses, and expression levels of these proteins were higher during grain filling than in the late stage of maturing. Localization of these proteins in the ER was confirmed by fluorescent immunostaining of the immature caryopses. In mature grains, the five groups of PDI family proteins remained in the aleurone cells and the protein matrix of the starchy endosperm.ConclusionsHigh expression of PDI family proteins during grain filling in the starchy endosperm suggest that these proteins play an important role in forming intramolecular disulfide bonds in seed storage proteins. In addition, these PDI family proteins that remain in the aleurone layers of mature grains likely assist in folding newly synthesized hydrolytic enzymes during germination.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0460-2) contains supplementary material, which is available to authorized users.
“…These hydrolytic enzymes are secreted to the starchy endosperm to break down starch and protein that accumulated in the starchy endosperm to supply carbon and nitrogen used in germination. Whereas the secretion of α-amylases and cysteine proteases from the aleurone layers increased in response to gibberellic acid treatment for 24 h, the level of the barley TaPDIL-1 ortholog was not affected by gibberellic acid treatment [64], suggesting that the barley TaPDIL-1 ortholog that remains in the aleurone cell of the wheat grain may help fold the newly synthesized hydrolytic enzymes.Figure 8
Distribution of wheat PDI family proteins in the mature aleurone cells and the protein matrix of wheat grains. Cross sections of mature grains were immunostained with sera against TaPDIL1Aα (A, F, K) , TaPDIL2 (B, G, L) , TaPDIL3A (C, H, M) , TaPDIL4D (D, I, N) , or TaPDIL5A (E, J, O) .…”
BackgroundThe major wheat seed proteins are storage proteins that are synthesized in the rough endoplasmic reticulum (ER) of starchy endosperm cells. Many of these proteins have intra- and intermolecular disulfide bonds. In eukaryotes, the formation of most intramolecular disulfide bonds in the ER is thought to be catalyzed by protein disulfide isomerase (PDI) family proteins. The cDNAs that encode eight groups of bread wheat (Triticum aestivum L.) PDI family proteins have been cloned, and their expression levels in developing wheat grains have been determined. The purpose of the present study was to characterize the enzymatic properties of the wheat PDI family proteins and clarify their expression patterns in wheat caryopses.ResultsPDI family cDNAs, which are categorized into group I (TaPDIL1Aα, TaPDIL1Aβ, TaPDIL1Aγ, TaPDIL1Aδ, and TaPDIL1B), group II (TaPDIL2), group III (TaPDIL3A), group IV (TaPDIL4D), and group V (TaPDIL5A), were cloned. The expression levels of recombinant TaPDIL1Aα, TaPDIL1B, TaPDIL2, TaPDIL3A, TaPDIL4D, and TaPDIL5A in Escherichia coli were established from the cloned cDNAs. All recombinant proteins were expressed in soluble forms and purified. Aside from TaPDIL3A, the recombinant proteins exhibited oxidative refolding activity on reduced and denatured ribonuclease A. Five groups of PDI family proteins were distributed throughout wheat caryopses, and expression levels of these proteins were higher during grain filling than in the late stage of maturing. Localization of these proteins in the ER was confirmed by fluorescent immunostaining of the immature caryopses. In mature grains, the five groups of PDI family proteins remained in the aleurone cells and the protein matrix of the starchy endosperm.ConclusionsHigh expression of PDI family proteins during grain filling in the starchy endosperm suggest that these proteins play an important role in forming intramolecular disulfide bonds in seed storage proteins. In addition, these PDI family proteins that remain in the aleurone layers of mature grains likely assist in folding newly synthesized hydrolytic enzymes during germination.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0460-2) contains supplementary material, which is available to authorized users.
“…Interestingly, we could explicitly identify more than 10 Golgi-resident proteins such as cell wall synthesis-associated proteins (e.g., UDP-glucuronic acid decarboxylase, A0A287H8Z0), which are parts of the glycosylation processes (Supplemental Table 2). It is possible that these identified proteins are active in aleurone, as previous data characterized the barley aleurone N-glycoproteome, in which numerous N-glycosylation sites were identified that play key roles in protein processing and secretion 73 .…”
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. During development, protein trafficking and storage require an extensive reorganization of the endomembrane system. Consequently, endomembrane-modifying proteins will influence the final grain quality and yield. However, little is known about the molecular mechanism underlying endomembrane system remodeling during barley grain development. By using label-free quantitative proteomics profiling, we quantified 1,822 proteins across developing barley grains. Based on proteome annotation and a homology search, 94 proteins associated with the endomembrane system were identified that exhibited significant changes in abundance during grain development. Clustering analysis allowed characterization of three different development phases; notably, integration of proteomics data with in situ subcellular microscopic analyses showed a high abundance of cytoskeleton proteins associated with acidified PBs at the early development stages. Moreover, 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 PBs during barley endosperm development. Together our data identified promising targets to be genetically engineered to modulate seed storage protein accumulation that have a growing role in health and nutritional issues. After differentiation, fully developed cereal endosperm makes up to 75% of the grain weight and covers four major cell types: aleurone, starchy endosperm, transfer cells, and the cells of the embryo surrounding region 1. The starchy endosperm thereby is characterized as a storage site, accumulating starch and seed storage proteins (SSPs) 2. The aleurone layer plays essential roles during seed germination and mobilizes starch and SSP reserves in the starchy endosperm by releasing hydrolytic enzymes that are responsible for the degradation of stored nutrients in the endosperm 2. Contrary to the persistent endosperm of cereals, the cellular endosperm of Arabidopsis thaliana (A. thaliana) supports the developing and growing embryo, resulting in a gradually depleted endosperm as the embryo grows. Finally, the massive A. thaliana embryo is only accompanied by a single peripheral layer, the aleurone layer, in mature seeds 2. Consequently, A. thaliana cannot to be used as a model system to study the endomembrane system in grain endosperm.
“…Tibbot et al [ 17 ] observed that the principal proteins in endosperm recognised by an Agl97 antiserum were of 101 and 95 kDa at 4 dpi, but of only 81 kDa two days later. Other possible modifications include variable levels of glycosylation and non-enzymic glycation, both of which have been proposed to account for the occurrence of multiple forms of other endosperm proteins [ 37 , 38 ].…”
During germination and early seedling growth of barley (Hordeum vulgare), maltase is responsible for the conversion of maltose produced by starch degradation in the endosperm to glucose for seedling growth. Despite the potential relevance of this enzyme for malting and the production of alcoholic beverages, neither the nature nor the role of maltase is fully understood. Although only one gene encoding maltase has been identified with certainty, there is evidence for the existence of other genes and for multiple forms of the enzyme. It has been proposed that maltase may be involved directly in starch granule degradation as well as in maltose hydrolysis. The aim of our work was to discover the nature of maltase in barley endosperm. We used ion exchange chromatography to fractionate maltase activity from endosperm of young seedlings, and we partially purified activity for protein identification. We compared maltase activity in wild-type barley and transgenic lines with reduced expression of the previously-characterised maltase gene Agl97, and we used genomic and transcriptomic information to search for further maltase genes. We show that all of the maltase activity in the barley endosperm can be accounted for by a single gene, Agl97. Multiple forms of the enzyme most likely arise from proteolysis and other post-translational modifications.
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