Global analysis of lysine acetylation in soybean leaves.
Scientific reports
Protein lysine acetylation (Kac) is an important post-translational modification in both animal and plant cells. Global Kac identification has been performed at the proteomic level in various species. However, the study of Kac in oil and resource plant species is relatively limited. Soybean is a globally important oil crop and resouce plant. In the present study, lysine acetylome analysis was performed in soybean leaves with proteomics techniques. Various bioinformatics analyses were performed to illustrate the structure and function of these Kac sites and proteins. Totally, 3148 acetylation sites in 1538 proteins were detected. Motif analysis of these Kac modified peptides extracted 17 conserved motifs. These Kac modified protein showed a wide subcellular location and functional distribution. Chloroplast is the primary subcellular location and cellular component where Kac proteins were localized. Function and pathways analyses indicated a plenty of biological processes and metabolism pathways potentially be influenced by Kac modification. Ribosome activity and protein biosynthesis, carbohydrate and energy metabolism, photosynthesis and fatty acid metabolism may be regulated by Kac modification in soybean leaves. Our study suggests Kac plays an important role in soybean physiology and biology, which is an available resource and reference of Kac function and structure characterization in oil crop and resource plant, as well as in plant kingdom.
10.1038/s41598-021-97338-9
N-linked glycoproteome analysis reveals central glycosylated proteins involved in wheat early seedling growth.
Wang Xueqian,Deng Xiong,Zhu Dong,Duan Wenjing,Zhang Junwei,Yan Yueming
Plant physiology and biochemistry : PPB
Glycosylation is an important protein post-translational modification in eukaryotic organisms. It is involved in many important life processes, such as cell recognition, differentiation, development, signal transduction and immune response. This study carried out the first N-linked glycosylation proteome analysis of wheat seedling leaves using HILIC glycosylation enrichment, chemical deglycosylation, HPLC separation and tandem mass spectrometric identification. In total, we detected 308 glycosylated peptides and 316 glycosylated sites corresponding to 248 unique glycoproteins. The identified glycoproteins were mainly concentrated in plasma membranes (25.6%), cell wall (16.8%) and extracellular area (16%). In terms of molecular function, 65% glycoproteins belonged to various enzymes with catalytic activity such as kinase, carboxypeptidase, peroxidase and phosphatase, and, particularly, 25% of glycoproteins were related to binding functions. These glycoproteins are involved in cell wall reconstruction, biomacromolecular metabolism, signal transduction, endoplasmic reticulum quality control and stress response. Analysis indicated that 57.66% of glycoproteins were highly conserved in other plant species while 42.34% of glycoproteins went unidentified among the conserved glycosylated homologous proteins in other plant species; these may be the new N-linked glycosylated proteins first identified in wheat. The glycosylation sites generally occurred on the random coil, which could play roles in maintaining the structural stability of proteins. PNGase F digestion and glycosylation site mutations further verified the glycosylation modification and glycosylation sites of LRR receptor-like serine/threonine-protein kinase (LRR-RLK) and Beta-D-glucan exohydrolase (β-D-GEH). Our results indicated that N-linked glycosylated proteins could play important roles in the early seedling growth of wheat.
10.1016/j.plaphy.2021.04.009
A comprehensive catalog of the lysine-acetylation targets in rice (Oryza sativa) based on proteomic analyses.
Xiong Yehui,Peng Xiaojun,Cheng Zhongyi,Liu Wende,Wang Guo-Liang
Journal of proteomics
Lysine acetylation is a dynamic and reversible post-translational modification that plays an important role in the gene transcription regulation. Here, we report high quality proteome-scale data for lysine-acetylation (Kac) sites and Kac proteins in rice (Oryza sativa). A total of 1337 Kac sites in 716 Kac proteins with diverse biological functions and subcellular localizations were identified in rice seedlings. About 42% of the sites were predicted to be localized in the chloroplast. Seven putative acetylation motifs were detected. Phenylalanine, located in both the upstream and downstream of the Kac sites, is the most conserved amino acid surrounding the regions. In addition, protein interaction network analysis revealed that a variety of signaling pathways are modulated by protein acetylation. KEGG pathway category enrichment analysis indicated that glyoxylate and dicarboxylate metabolism, carbon metabolism, and photosynthesis pathways are significantly enriched. Our results provide an in-depth understanding of the acetylome in rice seedlings, and the method described here will facilitate the systematic study of how Kac functions in growth, development, and abiotic and biotic stress responses in rice and other plants. BIOLOGICAL SIGNIFICANCE:Rice is one of the most important crops consumption and is a model monocot for research. In this study, we combined a highly sensitive immune-affinity purification method (used pan anti-acetyl-lysine antibody conjugated agarose for immunoaffinity acetylated peptide enrichment) with high-resolution LC-MS/MS. In total, we identified 1337 Kac sites on 716 Kac proteins in rice cells. Bioinformatic analysis of the acetylome revealed that the acetylated proteins are involved in a variety of cellular functions and have diverse subcellular localizations. We also identified seven putative acetylation motifs in the acetylated proteins of rice. In addition, protein interaction network analysis revealed that a variety of signaling pathways were modulated by protein acetylation. KEGG pathway category enrichment analysis indicated that glyoxylate and dicarboxylate metabolism, carbon metabolism, and photosynthesis pathways were significantly enriched. To our knowledge, the number of Kac sites we identified was 23-times greater and the number of Kac proteins was 16-times greater than in a previous report. Our results provide an in-depth understanding of the acetylome in rice seedlings, and the method described here will facilitate the systematic study of how Kac functions in growth, development and responses to abiotic and biotic stresses in rice or other plants.
10.1016/j.jprot.2016.01.019
First comprehensive proteome analysis of lysine crotonylation in seedling leaves of Nicotiana tabacum.
Scientific reports
Histone crotonylation is a new lysine acylation type of post-translational modification (PTM) enriched at active gene promoters and potential enhancers in yeast and mammalian cells. However, lysine crotonylation in nonhistone proteins and plant cells has not yet been studied. In the present study, we performed a global crotonylation proteome analysis of Nicotiana tabacum (tobacco) using high-resolution LC-MS/MS coupled with highly sensitive immune-affinity purification. A total of 2044 lysine modification sites distributed on 637 proteins were identified, representing the most abundant lysine acylation proteome reported in the plant kingdom. Similar to lysine acetylation and succinylation in plants, lysine crotonylation was related to multiple metabolism pathways, such as carbon metabolism, the citrate cycle, glycolysis, and the biosynthesis of amino acids. Importantly, 72 proteins participated in multiple processes of photosynthesis, and most of the enzymes involved in chlorophyll synthesis were modified through crotonylation. Numerous crotonylated proteins were implicated in the biosynthesis, folding, and degradation of proteins through the ubiquitin-proteasome system. Several crotonylated proteins related to chromatin organization are also discussed here. These data represent the first report of a global crotonylation proteome and provide a promising starting point for further functional research of crotonylation in nonhistone proteins.
10.1038/s41598-017-03369-6
A comparative glycoproteome study of developing endosperm in the hexose-deficient miniature1 (mn1) seed mutant and its wild type Mn1 in maize.
Silva-Sanchez Cecilia,Chen Sixue,Li Jinxi,Chourey Prem S
Frontiers in plant science
In maize developing seeds, transfer cells are prominently located at the basal endosperm transfer layer (BETL). As the first filial cell layer, BETL is a gateway to sugars, nutrients and water from mother plant; and anchor of numerous functions such as sucrose turnover, auxin and cytokinin biosynthesis/accumulation, energy metabolism, defense response, and signaling between maternal and filial generations. Previous studies showed that basal developing endosperms of miniature1 (mn1) mutant seeds lacking the Mn1-encoded cell wall invertase II, are also deficient for hexose. Given the role of glucose as one of the key sugars in protein glycosylation and proper protein folding; we performed a comparative large scale glycoproteome profiling of total proteins of these two genotypes (mn1 mutant vs. Mn1 wild type) using 2D gel electrophoresis and glycosylation/total protein staining, followed by image analysis. Protein identification was done by LC-MS/MS. A total of 413 spots were detected; from which, 113 spots matched between the two genotypes. Of these, 45 showed >20% decrease/increase in glycosylation level and were selected for protein identification. A large number of identified proteins showed decreased glycosylation levels in mn1 developing endosperms as compared to the Mn1. Functional classification of proteins, showed mainly of post-translational modification, protein turnover, chaperone activities, carbohydrate and amino acid biosynthesis/transport, and cell wall biosynthesis. These proteins and activities were related to endoplasmic reticulum (ER) stress and unfolded protein response (UPR) as a result of the low glycolsylation levels of the mutant proteins. Overall, these results provide for the first time a global glycoproteome profile of maize BETL-enriched basal endosperm to better understand their role in seed development in maize.
10.3389/fpls.2014.00063
Comprehensive profiling of the rice ubiquitome reveals the significance of lysine ubiquitination in young leaves.
Xie Xin,Kang Houxiang,Liu Wende,Wang Guo-Liang
Journal of proteome research
Protein ubiquitination is a major post-translational modification that regulates development, apoptosis, responses to environmental cues, and other processes in eukaryotes. Although several ubiquitinated proteins have been identified in rice, large-scale profiling of the rice ubiquitome has not been reported because of limitations in the current analytical methods. Here, we report the first rice ubiquitome, determined by combining highly sensitive immune affinity purification and high resolution LC-MS/MS. We identified 861 di-Gly-Lys-containing peptides in 464 proteins in rice leaf cells. Bioinformatic analyses of the ubiquitome identified a variety of cellular functions and diverse subcellular localizations for the ubiquitinated proteins, and also revealed seven putative ubiquitination motifs in rice. Proteins related to binding and catalytic activity were predicted to be the preferential targets of lysine ubiquitination. A protein interaction network and KEGG analysis indicated that a wide range of signaling and metabolic pathways are modulated by protein ubiquitination in rice. Our results demonstrate the usefulness of the significantly improved method for assaying proteome-wide ubiquitination in plants. The identification of the 464 ubiquitinated proteins in rice leaves provides a foundation for the analysis of the physiological roles of these ubiquitination-related proteins.
10.1021/pr5009724
Comparison of Antioxidant Constituents of Agriophyllum squarrosum Seed with Conventional Crop Seeds.
Journal of food science
Twelve chemical constituents were identified from the Agriophyllum squarrosum seed (ASS). ASS contained large amounts of flavonoids, which were more concentrated in the seed coat. ASS-coat (1 g) contained 335.7 μg flavonoids of rutin equivalent, which was similar to the flavonoid content in soybean (351.2 μg/g), and greater than that in millet, wheat, rice, peanut, and corn. By LC-MS analysis, the major constituents in ASS were 3-O-[α-L-rhamnopyranosyl-(1→6)-β-D- glucopyranosyl]-7- O-(β-D-glucopyranosyl)-quercetin (1), rutin (4), quercetin-3-O-β-D- apiosyl(1→2)-[α-L-rhamnosyl(l→6)]-β-D-glucoside (2), isorhamnetin-3-O-rutinoside (5), and allantoin (3), compared with isoflavonoids-genistin (16), daidzin (14), and glycitin (18) in soybean. Among constituents in ASS, compounds 1, 2, 4, protocatechuic acid (8), isoquercitrin (11), and luteolin-6-C-glucoside (12) potently scavenged DPPH radicals and intracellular ROS; strongly protected against peroxyl radical-induced DNA scission; and upregulated Nrf2, phosphorylated p38, phosphorylated JNK, and Bcl-2 in HepG2 cells. These results indicate that ASS is rich in antioxidant constituents that can enrich the varieties of food flavonoids, with significant beneficial implications for those who suffer from oxidative stress-related conditions. PRACTICAL APPLICATION:This study found that A. squarrosum seed contains large amounts of antioxidative flavonoids and compared its chemical constituents with those of conventional foods. These results should increase the interest in planting the sand-fixing A. squarrosum on a large scale, thus preventing desertification and providing valuable foods.
10.1111/1750-3841.14159
Mass spectrometric resolution of reversible protein phosphorylation in photosynthetic membranes of Arabidopsis thaliana.
Vener A V,Harms A,Sussman M R,Vierstra R D
The Journal of biological chemistry
The use of mass spectrometry to characterize the phosphorylome, i.e. the constituents of the proteome that become phosphorylated, was demonstrated using the reversible phosphorylation of chloroplast thylakoid proteins as an example. From the analysis of tryptic peptides released from the surface of Arabidopsis thylakoids, the principal phosphoproteins were identified by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry. These studies revealed that the D1, D2, and CP43 proteins of the photosystem II core are phosphorylated at their N-terminal threonines (Thr), the peripheral PsbH protein is phosphorylated at Thr-2, and the mature light-harvesting polypeptides LCHII are phosphorylated at Thr-3. In addition, a doubly phosphorylated form of PsbH modified at both Thr-2 and Thr-4 was detected. By comparing the levels of phospho- and nonphosphopeptides, the in vivo phosphorylation states of these proteins were analyzed under different physiological conditions. None of these thylakoid proteins were completely phosphorylated in the steady state conditions of continuous light or completely dephosphorylated after a long dark adaptation. However, rapid reversible hyperphosphorylation of PsbH at Thr-4 in response to growth in light/dark transitions and a pronounced specific dephosphorylation of the D1, D2, and CP43 proteins during heat shock was detected. Collectively, our data indicate that changes in the phosphorylation of photosynthetic proteins are more rapid during heat stress than during normal light/dark transitions. These mass spectrometry methods offer a new approach to assess the stoichiometry of in vivo protein phosphorylation in complex samples.
10.1074/jbc.M009394200
Quantitative Circadian Phosphoproteomic Analysis of Arabidopsis Reveals Extensive Clock Control of Key Components in Physiological, Metabolic, and Signaling Pathways.
Choudhary Mani Kant,Nomura Yuko,Wang Lei,Nakagami Hirofumi,Somers David E
Molecular & cellular proteomics : MCP
The circadian clock provides adaptive advantages to an organism, resulting in increased fitness and survival. The phosphorylation events that regulate circadian-dependent signaling and the processes which post-translationally respond to clock-gated signals are largely unknown. To better elucidate post-translational events tied to the circadian system we carried out a survey of circadian-regulated protein phosphorylation events in Arabidopsis seedlings. A large-scale mass spectrometry-based quantitative phosphoproteomics approach employing TiO2-based phosphopeptide enrichment techniques identified and quantified 1586 phosphopeptides on 1080 protein groups. A total of 102 phosphopeptides displayed significant changes in abundance, enabling the identification of specific patterns of response to circadian rhythms. Our approach was sensitive enough to quantitate oscillations in the phosphorylation of low abundance clock proteins (early flowering4; ELF4 and pseudoresponse regulator3; PRR3) as well as other transcription factors and kinases. During constant light, extensive cyclic changes in phosphorylation status occurred in critical regulators, implicating direct or indirect regulation by the circadian system. These included proteins influencing transcriptional regulation, translation, metabolism, stress and phytohormones-mediated responses. We validated our analysis using the elf4-211 allele, in which an S45L transition removes the phosphorylation herein identified. We show that removal of this phosphorylatable site diminishes interaction with early flowering3 (ELF3), a key partner in a tripartite evening complex required for circadian cycling. elf4-211 lengthens period, which increases with increasing temperature, relative to the wild type, resulting in a more stable temperature compensation of circadian period over a wider temperature range.
10.1074/mcp.M114.047183
A phyloproteomic characterization of in vitro autophosphorylation in calcium-dependent protein kinases.
Hegeman Adrian D,Rodriguez Miguel,Han Byung Woo,Uno Yuichi,Phillips George N,Hrabak Estelle M,Cushman John C,Harper Jeff F,Harmon Alice C,Sussman Michael R
Proteomics
Calcium-dependent protein kinases (CDPKs) are a novel class of signaling molecules that have been broadly implicated in relaying specific calcium-mediated responses to biotic and abiotic stress as well as developmental cues in both plants and protists. Calcium-dependent autophosphorylation has been observed in almost all CDPKs examined, but a physiological role for autophosphorylation has not been demonstrated. To date, only a handful of autophosphorylation sites have been mapped to specific residues within CDPK amino acid sequences. In an attempt to gain further insight into this phenomenon, we have mapped autophosphorylation sites and compared these phosphorylation patterns among multiple CDPK isoforms. From eight CDPKs and two CDPK-related kinases from Arabidopsis thaliana and Plasmodium falciparum, 31 new autophosphorylation sites were characterized, which in addition to the previously described sites, allowed the identification of five conserved loci. Of the 35 total sites analyzed approximately one-half were observed in the N-terminal variable domain. Homology models were generated for the protein kinase and calmodulin-like domains, each containing two of the five conserved sites, to allow intelligent speculation regarding subsequent lines of investigation.
10.1002/pmic.200500926
Lysine acetylome profiling uncovers novel histone deacetylase substrate proteins in .
Hartl Markus,Füßl Magdalena,Boersema Paul J,Jost Jan-Oliver,Kramer Katharina,Bakirbas Ahmet,Sindlinger Julia,Plöchinger Magdalena,Leister Dario,Uhrig Glen,Moorhead Greg Bg,Cox Jürgen,Salvucci Michael E,Schwarzer Dirk,Mann Matthias,Finkemeier Iris
Molecular systems biology
Histone deacetylases have central functions in regulating stress defenses and development in plants. However, the knowledge about the deacetylase functions is largely limited to histones, although these enzymes were found in diverse subcellular compartments. In this study, we determined the proteome-wide signatures of the RPD3/HDA1 class of histone deacetylases in Relative quantification of the changes in the lysine acetylation levels was determined on a proteome-wide scale after treatment of leaves with deacetylase inhibitors apicidin and trichostatin A. We identified 91 new acetylated candidate proteins other than histones, which are potential substrates of the RPD3/HDA1-like histone deacetylases in , of which at least 30 of these proteins function in nucleic acid binding. Furthermore, our analysis revealed that histone deacetylase 14 (HDA14) is the first organellar-localized RPD3/HDA1 class protein found to reside in the chloroplasts and that the majority of its protein targets have functions in photosynthesis. Finally, the analysis of HDA14 loss-of-function mutants revealed that the activation state of RuBisCO is controlled by lysine acetylation of RuBisCO activase under low-light conditions.
10.15252/msb.20177819
Multiplex staining of 2-DE gels for an initial phosphoproteome analysis of germinating seeds and early grown seedlings from a non-orthodox specie: Quercus ilex L. subsp. ballota [Desf.] Samp.
Romero-Rodríguez M Cristina,Abril Nieves,Sánchez-Lucas Rosa,Jorrín-Novo Jesús V
Frontiers in plant science
As a preliminary step in the phosphoproteome analysis of germinating seeds (0 and 24 h after seed imbibition) and early grown seedlings (216 h after seed imbibition) from a non-orthodox sp. Quercus ilex, a multiplex (SYPRO-Ruby and Pro-Q DPS) staining of high-resolution 2-DE gels was used. By using this protocol it was possible to detect changes in protein-abundance and/or phosphorylation status. This simple approach could be a good complementary alternative to the enrichment protocols used in the search for phosphoprotein candidates. While 482 spots were visualized with SYPRO-Ruby, 222 were with Pro-Q DPS. Statistically significant differences in spot intensity were observed among samples, these corresponding to 85 SYPRO-Ruby-, 20 Pro-Q-DPS-, and 35 SYPRO-Ruby and Pro-Q-DPS-stained spots. Fifty-five phosphoprotein candidates showing qualitative or quantitative differences between samples were subjected to MALDI-TOF-TOF MS analysis, with 20 of them being identified. Identified proteins belonged to five different functional categories, namely: carbohydrate and amino acid metabolism, defense, protein folding, and oxidation-reduction processes. With the exception of a putative cyclase, the other 19 proteins had at least one orthologous phosphoprotein in Arabidopsis thaliana, Medicago truncatula, N. tabacum, and Glycine max. Out of the 20 identified, seven showed differences in intensity in Pro-Q-DPS but not in SYPRO-Ruby-stained gels, including enzymes of the glycolysis and amino acid metabolism. This bears out that theory the regulation of these enzymes occurs at the post-translational level by phosphorylation with no changes at the transcriptional or translational level. This is different from the mechanism reported in orthodox seeds, in which concomitant changes in abundance and phosphorylation status have been observed for these enzymes.
10.3389/fpls.2015.00620