Post-translational Modifications In Plants Research Articles

Protein pyrophosphorylation by inositol phosphates: a novel post-translational modification in plants?

Inositol pyrophosphates (PP-InsPs) are energy-rich molecules harboring one or more diphosphate moieties. PP-InsPs are found in all eukaryotes evaluated and their functional versatility is reflected in the various cellular events in which they take part. These include, among others, insulin signaling and intracellular trafficking in mammals, as well as innate immunity and hormone and phosphate signaling in plants. The molecular mechanisms by which PP-InsPs exert such functions are proposed to rely on the allosteric regulation via direct binding to proteins, by competing with other ligands, or by protein pyrophosphorylation. The latter is the focus of this review, where we outline a historical perspective surrounding the first findings, almost 20 years ago, that certain proteins can be phosphorylated by PP-InsPs in vitro. Strikingly, in vitro phosphorylation occurs by an apparent enzyme-independent but Mg2+-dependent transfer of the β-phosphoryl group of an inositol pyrophosphate to an already phosphorylated serine residue at Glu/Asp-rich protein regions. Ribosome biogenesis, vesicle trafficking and transcription are among the cellular events suggested to be modulated by protein pyrophosphorylation in yeast and mammals. Here we discuss the latest efforts in identifying targets of protein pyrophosphorylation, pointing out the methodological challenges that have hindered the full understanding of this unique post-translational modification, and focusing on the latest advances in mass spectrometry that finally provided convincing evidence that PP-InsP-mediated pyrophosphorylation also occurs in vivo. We also speculate about the relevance of this post-translational modification in plants in a discussion centered around the protein kinase CK2, whose activity is critical for pyrophosphorylation of animal and yeast proteins. This enzyme is widely present in plant species and several of its functions overlap with those of PP-InsPs. Until now, there is virtually no data on pyrophosphorylation of plant proteins, which is an exciting field that remains to be explored.

N-Linked Glycoproteome Analysis of Diosorea alata Tuber Shows Atypical Glycosylation and Indicates Central Role of Glycosylated Proteins in Tuber Maturation.

Glycosylation is an important post translational modification in plants. First analysis of N-linked glycosylated proteins of Dioscorea alata using Concanavalin A lectin affinity chromatography enrichment coupled with label free quantification is presented. In total, 114 enriched glycoproteins were detected. Signal P and sub-cellular localization showed 42.2% of proteins to be secretory. These included peroxidases, endochitinases, calreticulin, calnexin, thaumatins and lipid transfer proteins. Gene Ontology and MapMan analysis predicted the enriched glycoproteins to be involved in processes essential for tuber maturation namely: signal transduction, lignification, protein trafficking, endoplasmic reticulum quality control and cell wall remodeling. This was supported by biochemical validation of the essential glycoproteins. Interestingly, out of the two dioscorin isoforms, Dio B was the only N-glycosylated form. In silico analysis showed O-glycosylation sites in the other form, Dio A suggesting its similarity with sporamin, the storage protein of sweet potato. Absence of signal peptide in Dio B and the presence of non-canonical motif hints towards its atypical glycosylation. The analysis revealed that N-glycosylation of Dio B isoform maintains the activities associated with Dioscorin at maturity and provides an overview of protein N-glycosylation, enriching the glycoproteome database of plants especially tubers.

An atlas of Arabidopsis protein S-acylation reveals its widespread role in plant cell organization and function.

S-acylation is the addition of a fatty acid to a cysteine residue of a protein. While this modification may profoundly alter protein behaviour, its effects on the function of plant proteins remains poorly characterized, largely as a result of the lack of basic information regarding which proteins are S-acylated and where in the proteins the modification occurs. To address this gap in our knowledge, we used an optimized acyl-resin-assisted capture assay to perform a comprehensive analysis of plant protein S-acylation from six separate tissues. In our high- and medium-confidence groups, we identified 1,849 cysteines modified by S-acylation, which were located in 1,640 unique peptides from 1,094 different proteins. This represents around 6% of the detectable Arabidopsis proteome and suggests an important role for S-acylation in many essential cellular functions including trafficking, signalling and metabolism. To illustrate the potential of this dataset, we focus on cellulose synthesis and confirm the S-acylation of a number of proteins known to be involved in cellulose synthesis and trafficking of the cellulose synthase complex. In the secondary cell walls, cellulose synthesis requires three different catalytic subunits (CESA4, CESA7 and CESA8) that all exhibit striking sequence similarity and are all predicted to possess a RING-type zinc finger at their amino terminus composed of eight cysteines. For CESA8, we find evidence for S-acylation of these cysteines that is incompatible with any role in coordinating metal ions. We show that while CESA7 may possess a RING-type domain, the same region of CESA8 appears to have evolved a very different structure. Together, the data suggest that this study represents an atlas of S-acylation in Arabidopsis that will facilitate the broader study of this elusive post-translational modification in plants as well as demonstrating the importance of further work in this area.

Spatial Mapping of Plant N-Glycosylation Cellular Heterogeneity Inside Soybean Root Nodules Provided Insights Into Legume-Rhizobia Symbiosis.

Although ubiquitously present, information on the function of complex N-glycan posttranslational modification in plants is very limited and is often neglected. In this work, we adopted an enzyme-assisted matrix-assisted laser desorption/ionization mass spectrometry imaging strategy to visualize the distribution and identity of N-glycans in soybean root nodules at a cellular resolution. We additionally performed proteomics analysis to probe the potential correlation to proteome changes during symbiotic rhizobia-legume interactions. Our ion images reveal that intense N-glycosylation occurs in the sclerenchyma layer, and inside the infected cells within the infection zone, while morphological structures such as the cortex, uninfected cells, and cells that form the attachment with the root are fewer N-glycosylated. Notably, we observed different N-glycan profiles between soybean root nodules infected with wild-type rhizobia and those infected with mutant rhizobia incapable of efficiently fixing atmospheric nitrogen. The majority of complex N-glycan structures, particularly those with characteristic Lewis-a epitopes, are more abundant in the mutant nodules. Our proteomic results revealed that these glycans likely originated from proteins that maintain the redox balance crucial for proper nitrogen fixation, but also from enzymes involved in N-glycan and phenylpropanoid biosynthesis. These findings indicate the possible involvement of Lewis-a glycans in these critical pathways during legume-rhizobia symbiosis.

Proteome-wide identification of non-histone lysine methylation in tomato during fruit ripening

IntroductionHistone and non-histone methylations are important post-translational modifications in plants. Histone methylation plays a crucial role in regulating chromatin structure and gene expression. However, the involvement of non-histone methylation in plant biological processes remains largely unknown. MethodsThe methylated substrates and methylation sites during tomato fruit ripening were identified by LC-MS/MS. Bioinformatics of lysine methylated proteins was conducted to analyze the possible role of methylated proteins. The effects of methylation modification on protein functions were preliminarily investigated by site-directed mutation simulation. ResultsA total of 241 lysine methylation (mono-, di- and trimethylation) sites in 176 proteins were identified with two conserved methylation motifs: xxxxxxExxx_K_xxxExxxxxx and xxxxxxExxx_K_xxxxxxxxxx. These methylated proteins were mainly related to fruit ripening and senescence, oxidation reduction process, signal transduction, stimulus and stress responses, and energy metabolism. Three representative proteins, thioredoxin (Trx), glutathione S-transferase T1 (GST T1), and NADH dehydrogenase (NOX), were selected to investigate the effect of methylation modifications on protein activity. Mimicking demethylation led to decreased Trx activity but increased GST T1 and NOX activities. In addition, RT-qPCR exhibited that the expression of many genes that encode proteins subjected to methylation was upregulated during fruit ripening. ConclusionOur study suggests that tomato fruit ripening undergo non-histone lysine methylation, which may participate in the regulation of fruit ripening. It is the first report of methyl proteome profiling of non-histone lysine in horticultural crops.

(De)Activation (Ir)Reversibly or Degradation: Dynamics of Post-Translational Protein Modifications in Plants.

The increasing dynamic functions of post-translational modifications (PTMs) within protein molecules present outstanding challenges for plant biology even at this present day. Protein PTMs are among the first and fastest plant responses to changes in the environment, indicating that the mechanisms and dynamics of PTMs are an essential area of plant biology. Besides being key players in signaling, PTMs play vital roles in gene expression, gene, and protein localization, protein stability and interactions, as well as enzyme kinetics. In this review, we take a broader but concise approach to capture the current state of events in the field of plant PTMs. We discuss protein modifications including citrullination, glycosylation, phosphorylation, oxidation and disulfide bridges, N-terminal, SUMOylation, and ubiquitination. Further, we outline the complexity of studying PTMs in relation to compartmentalization and function. We conclude by challenging the proteomics community to engage in holistic approaches towards identification and characterizing multiple PTMs on the same protein, their interaction, and mechanism of regulation to bring a deeper understanding of protein function and regulation in plants.

QPTMplants: an integrative database of quantitative post-translational modifications in plants.

As a crucial molecular mechanism, post-translational modifications (PTMs) play critical roles in a wide range of biological processes in plants. Recent advances in mass spectrometry-based proteomic technologies have greatly accelerated the profiling and quantification of plant PTM events. Although several databases have been constructed to store plant PTM data, a resource including more plant species and more PTM types with quantitative dynamics still remains to be developed. In this paper, we present an integrative database of quantitative PTMs in plants named qPTMplants (http://qptmplants.omicsbio.info), which hosts 1 242 365 experimentally identified PTM events for 429 821 nonredundant sites on 123 551 proteins under 583 conditions for 23 PTM types in 43 plant species from 293 published studies, with 620 509 quantification events for 136 700 PTM sites on 55 361 proteins under 354 conditions. Moreover, the experimental details, such as conditions, samples, instruments and methods, were manually curated, while a variety of annotations, including the sequence and structural characteristics, were integrated into qPTMplants. Then, various search and browse functions were implemented to access the qPTMplants data in a user-friendly manner. Overall, we anticipate that the qPTMplants database will be a valuable resource for further research on PTMs in plants.

In vivo homopropargylglycine incorporation enables sampling, isolation and characterization of nascent proteins from Arabidopsis thaliana.

Determining which proteins are actively synthesized at a given point in time and extracting a representative sample for analysis is important to understand plant responses. Here we show that the methionine (Met) analogue homopropargylglycine (HPG) enables Bio-Orthogonal Non-Canonical Amino acid Tagging (BONCAT) of a small sample of the proteins being synthesized in Arabidopsis plants or cell cultures, facilitating their click-chemistry enrichment for analysis. The sites of HPG incorporation could be confirmed by peptide mass spectrometry at Met sites throughout protein amino acid sequences and correlation with independent studies of protein labelling with 15 N verified the data. We provide evidence that HPG-based BONCAT tags a better sample of nascent plant proteins than azidohomoalanine (AHA)-based BONCAT in Arabidopsis and show that the AHA induction of Met metabolism and greater inhibition of cell growth rate than HPG probably limits AHA incorporation at Met sites in Arabidopsis. We show HPG-based BONCAT provides a verifiable method for sampling, which plant proteins are being synthesized at a given time point and enriches a small portion of new protein molecules from the bulk protein pool for identification, quantitation and subsequent biochemical analysis. Enriched nascent polypeptides samples were found to contain significantly fewer common post-translationally modified residues than the same proteins from whole plant extracts, providing evidence for age-related accumulation of post-translational modifications in plants.

C-terminal tyrosine removal from wheat low-molecular weight glutenin subunits (LMW-GS); biologically relevant or mistaken substrate?

By applying a high-resolution LC-MS method, proteoform profiling of wheat glutenin proteins has revealed numerous protein pairs differing by an average mass of 163 ± 1 Da. The most abundant of 21 such pairs, 40 344 Da and 40 181 Da (coded P40344 and P40181, respectively), were purified and subjected to peptide mapping. P40344 matched the calculated mature protein mass for Uniprot B2Y2Q7, an s-type low-molecular weight glutenin subunit (LMW-GS). The P40344 and P40181 digests differed by a single digest peptide, unambiguously matched to the C-terminus of the protein and differing by the loss of a tyrosine. Of 327 unique mature wheat LMW-GS sequences from 369 UNIPROT accessions, tyrosine is at the C-terminus in all but one accession with evidence of transcription. The C-terminal LMW-GS sequences are also highly conserved, with only 10 variants of the last six amino acids. This is the first observation of cleavage of a C-terminal tyrosine other than alpha-tubulin, and the first evidence of this post-translational modification in plants. The identification of this novel post-translational modifications in gluten proteins may provide new insights on the differential modulation of biological processes involved in seed maturation and their effect on functional properties.

Mass Spectrometry-Based Identification of Phospho-Tyr in Plant Proteomics.

O-Phosphorylation (phosphorylation of the hydroxyl-group of S, T, and Y residues) is among the first described and most thoroughly studied posttranslational modification (PTM). Y-Phosphorylation, catalyzed by Y-kinases, is a key step in both signal transduction and regulation of enzymatic activity in mammalian systems. Canonical Y-kinase sequences are absent from plant genomes/kinomes, often leading to the assumption that plant cells lack O-phospho-l-tyrosine (pY). However, recent improvements in sample preparation, coupled with advances in instrument sensitivity and accessibility, have led to results that unequivocally disproved this assumption. Identification of hundreds of pY-peptides/proteins, followed by validation using genomic, molecular, and biochemical approaches, implies previously unappreciated roles for this "animal PTM" in plants. Herein, we review extant results from studies of pY in plants and propose a strategy for preparation and analysis of pY-peptides that will allow a depth of coverage of the plant pY-proteome comparable to that achieved in mammalian systems.

In-depth characterization of Trichoderma reesei cellobiohydrolase TrCel7A produced in Nicotiana benthamiana reveals limitations of cellulase production in plants by host-specific post-translational modifications.

SummarySustainable production of biofuels from lignocellulose feedstocks depends on cheap enzymes for degradation of such biomass. Plants offer a safe and cost‐effective production platform for biopharmaceuticals, vaccines and industrial enzymes boosting biomass conversion to biofuels. Production of intact and functional protein is a prerequisite for large‐scale protein production, and extensive host‐specific post‐translational modifications (PTMs) often affect the catalytic properties and stability of recombinant enzymes. Here we investigated the impact of plant PTMs on enzyme performance and stability of the major cellobiohydrolase TrCel7A from Trichoderma reesei, an industrially relevant enzyme. TrCel7A was produced in Nicotiana benthamiana using a vacuum‐based transient expression technology, and this recombinant enzyme (TrCel7Arec) was compared with the native fungal enzyme (TrCel7Anat) in terms of PTMs and catalytic activity on commercial and industrial substrates. We show that the N‐terminal glutamate of TrCel7Arec was correctly processed by N. benthamiana to a pyroglutamate, critical for protein structure, while the linker region of TrCel7Arec was vulnerable to proteolytic digestion during protein production due to the absence of O‐mannosylation in the plant host as compared with the native protein. In general, the purified full‐length TrCel7Arec had 25% lower catalytic activity than TrCel7Anat and impaired substrate‐binding properties, which can be attributed to larger N‐glycans and lack of O‐glycans in TrCel7Arec. All in all, our study reveals that the glycosylation machinery of N. benthamiana needs tailoring to optimize the production of efficient cellulases.

The Plant PTM Viewer, a central resource for exploring plant protein modifications.

Post-translational modifications (PTMs) of proteins are central in any kind of cellular signaling. Modern mass spectrometry technologies enable comprehensive identification and quantification of various PTMs. Given the increased numbers and types of mapped protein modifications, a database is necessary that simultaneously integrates and compares site-specific information for different PTMs, especially in plants for which the available PTM data are poorly catalogued. Here, we present the Plant PTM Viewer (http://www.psb.ugent.be/PlantPTMViewer), an integrative PTM resource that comprises approximately 370000 PTM sites for 19 types of protein modifications in plant proteins from five different species. The Plant PTM Viewer provides the user with a protein sequence overview in which the experimentally evidenced PTMs are highlighted together with an estimate of the confidence by which the modified peptides and, if possible, the actual modification sites were identified and with functional protein domains or active site residues. The PTM sequence search tool can query PTM combinations in specific protein sequences, whereas the PTM BLAST tool searches for modified protein sequences to detect conserved PTMs in homologous sequences. Taken together, these tools help to assume the role and potential interplay of PTMs in specific proteins or within a broader systems biology context. The Plant PTM Viewer is an open repository thatallows the submission of mass spectrometry-based PTM data to remain at pace with future PTM plant studies.

Detection of S-Nitrosated Nuclear Proteins in Pathogen-Treated Arabidopsis Cell Cultures Using Biotin Switch Technique.

Nitric oxide (NO) is an important signaling molecule involved in various plant physiological processes. The main effect of NO arises from its reaction with proteins. S-Nitrosation is the most studied NO-mediated protein posttranslational modification in plants. Via S-nitrosation, NO derivatives react with thiol groups (SHs) of protein cysteine residues and produce nitrosothiol groups (SNOs). From the time of discovering the biological function of NO in plants, an interesting case of study has been the detection of the endogenous S-nitrosated proteins in different plants, tissues, organelles, and various conditions. Maps of S-nitrosated proteins provide hints for deeper studies on the function of this modification in specific proteins, biochemical pathways, and physiological processes. Many functions of NO have been found to be related to plant defense; on the other hand the involvement of nuclear proteins in regulation of plant defense reactions is well studied. Here, an approach is described in which the Arabidopsis cell cultures first are treated with P. syringae, afterward their bioactive nuclear proteins are extracted, then the nuclear proteins are subjected to biotin switch assay in which S-nitrosated proteins are specifically converted to S-biotinylated proteins. The biotin switch technique (BST) which was introduced by Jaffrey et al. (Nat Cell Biol 3:193-197, 2001) solves the instability issue of SNOs. Additionally, it provides detection and purification of biotinylated proteins by anti-biotin antibody and affinity chromatography, respectively.

Proteome-wide identification of lysine 2-hydroxyisobutyrylation reveals conserved and novel histone modifications in Physcomitrella patens

Protein lysine 2-hydroxyisobutyrylation (Khib) is a newly identified post-translational modification found in animal and yeast cells. Previous research suggested that histone Khib is involved in male cell differentiation and plays a critical role in the regulation of chromatin functions in animals. However, information regarding protein Khib in plants is still limited. In this study, using a specific antibody and LC-MS/MS methods, we identified 11,976 Khib sites in 3,001 proteins in Physcomitrella patens. The bioinformatics analysis indicated that these Khib-modified proteins were involved in a wide range of molecular functions and cellular processes, and showed diverse subcellular localizations. Furthermore, an comparism of Khib sites in histone proteins among human, mouse and P. patens found conserved sites in the H3 and H4 histone proteins and novel sites in H1, H2A and H2B histone proteins in P. patens. This is the first report on Khib post-translational modifications in plants, and the study provides a comprehensive profile of Khib sites in histone and non-histone proteins in Physcomitrella patens.

Up-to-Date Workflow for Plant (Phospho)proteomics Identifies Differential Drought-Responsive Phosphorylation Events in Maize Leaves.

Protein phosphorylation is one of the most common post-translational modifications (PTMs), which can regulate protein activity and localization as well as protein-protein interactions in numerous cellular processes. Phosphopeptide enrichment techniques enable plant researchers to acquire insight into phosphorylation-controlled signaling networks in various plant species. Most phosphoproteome analyses of plant samples still involve stable isotope labeling, peptide fractionation, and demand a lot of mass spectrometry (MS) time. Here, we present a simple workflow to probe, map, and catalogue plant phosphoproteomes, requiring relatively low amounts of starting material, no labeling, no fractionation, and no excessive analysis time. Following optimization of the different experimental steps on Arabidopsis thaliana samples, we transferred our workflow to maize, a major monocot crop, to study signaling upon drought stress. In addition, we included normalization to protein abundance to identify true phosphorylation changes. Overall, we identified a set of new phosphosites in both Arabidopsis thaliana and maize, some of which are differentially phosphorylated upon drought. All data are available via ProteomeXchange with identifier PXD003634, but to provide easy access to our model plant and crop data sets, we created an online database, Plant PTM Viewer ( bioinformatics.psb.ugent.be/webtools/ptm_viewer/ ), where all phosphosites identified in our study can be consulted.

DYn-2 Based Identification of Arabidopsis Sulfenomes*[S

Identifying the sulfenylation state of stressed cells is emerging as a strategic approach for the detection of key reactive oxygen species signaling proteins. Here, we optimized an in vivo trapping method for cysteine sulfenic acids in hydrogen peroxide (H2O2) stressed plant cells using a dimedone based DYn-2 probe. We demonstrated that DYn-2 specifically detects sulfenylation events in an H2O2 dose- and time-dependent way. With mass spectrometry, we identified 226 sulfenylated proteins after H2O2 treatment of Arabidopsis cells, residing in the cytoplasm (123); plastid (68); mitochondria (14); nucleus (10); endoplasmic reticulum, Golgi and plasma membrane (7) and peroxisomes (4). Of these, 123 sulfenylated proteins have never been reported before to undergo cysteine oxidative post-translational modifications in plants. All in all, with this DYn-2 approach, we have identified new sulfenylated proteins, and gave a first glance on the locations of the sulfenomes of Arabidopsis thaliana.

Protein S-nitrosylation: specificity and identification strategies in plants.

The role of nitric oxide (NO) as a major regulator of plant physiological functions has become increasingly evident. To further improve our understanding of its role, within the last few years plant biologists have begun to embrace the exciting opportunity of investigating protein S-nitrosylation, a major reversible NO-dependent post-translational modification (PTM) targeting specific Cys residues and widely studied in animals. Thanks to the development of dedicated proteomic approaches, in particular the use of the biotin switch technique (BST) combined with mass spectrometry, hundreds of plant protein candidates for S-nitrosylation have been identified. Functional studies focused on specific proteins provided preliminary comprehensive views of how this PTM impacts the structure and function of proteins and, more generally, of how NO might regulate biological plant processes. The aim of this review is to detail the basic principle of protein S-nitrosylation, to provide information on the biochemical and structural features of the S-nitrosylation sites and to describe the proteomic strategies adopted to investigate this PTM in plants. Limits of the current approaches and tomorrow's challenges are also discussed.

Gene Targeting for Precision Glyco-Engineering: Production of Biopharmaceuticals Devoid of Plant-Typical Glycosylation in Moss Bioreactors.

One of the main challenges for the production of biopharmaceuticals in plant-based systems is the modulation of plant-specific glycosylation patterns towards a humanized form. Posttranslational modifications in plants are similar to those in humans, but several differences affect product quality and efficacy and can also cause immune responses in patients. In the moss Physcomitrella patens highly efficient gene targeting via homologous recombination enables glyco-engineering to obtain suitable platform lines for the production of recombinant proteins and biopharmaceuticals. Here we describe the methods which are effective for creating gene targeting constructs and transgenic moss lines as well as confirming successful homologous integration of the constructs and modification of target gene expression.

Is Lys-Nɛ-acetylation the next big thing in post-translational modifications?

Lys-N(ɛ)-acetylation (PKA) has recently ascended from a post-translational modification (PTM) of limited distribution to one approaching the abundance of O-phosphorylation. Thousands of KAC proteins have been identified in Archaea, bacteria, and Eukarya, and the KAC system of acetyltransferases, deacetylases, and binding proteins is superficially comparable with the kinases, phosphatases, and phospho- (P-)protein binding-proteins of O-phosphorylation. Herein, we describe recent results and compare several aspects of these two major systems of PTM in plants.

Correlations between predicted protein disorder and post-translational modifications in plants

Motivation: Protein structural research in plants lags behind that in animal and bacterial species. This lag concerns both the structural analysis of individual proteins and the proteome-wide characterization of structure-related properties. Until now, no systematic study concerning the relationships between protein disorder and multiple post-translational modifications (PTMs) in plants has been presented.Results: In this work, we calculated the global degree of intrinsic disorder in the complete proteomes of eight typical monocotyledonous and dicotyledonous plant species. We further predicted multiple sites for phosphorylation, glycosylation, acetylation and methylation and examined the correlations of protein disorder with the presence of the predicted PTM sites. It was found that phosphorylation, acetylation and O-glycosylation displayed a clear preference for occurrence in disordered regions of plant proteins. In contrast, methylation tended to avoid disordered sequence, whereas N-glycosylation did not show a universal structural preference in monocotyledonous and dicotyledonous plants. In addition, the analysis performed revealed significant differences between the integral characteristics of monocot and dicot proteomes. They included elevated disorder degree, increased rate of O-glycosylation and R-methylation, decreased rate of N-glycosylation, K-acetylation and K-methylation in monocotyledonous plant species, as compared with dicotyledonous species. Altogether, our study provides the most compelling evidence so far for the connection between protein disorder and multiple PTMs in plants.Contact: tokmak@phoenix.kobe-u.ac.jp or tetsuya.sakurai@riken.jpSupplementary information: Supplementary data are available at Bioinformatics online.