Top-down mass spectrometry of native proteoforms and their complexes: a community study.

The combination of native electrospray ionization with top-down fragmentation in mass spectrometry (MS) allows simultaneous determination of the stoichiometry of noncovalent complexes and identification of their component proteoforms and cofactors. Although this approach is powerful, both native MS and top-down MS are not yet well standardized, and only a limited number of laboratories regularly carry out this type of research. To address this challenge, the Consortium for Top-Down Proteomics initiated a study to develop and test protocols for native MS combined with top-down fragmentation of proteins and protein complexes across 11 instruments in nine laboratories. Here we report the summary of the outcomes to provide robust benchmarks and a valuable entry point for the scientific community.

Spatially Resolved Top-Down Proteomics of Tissue Sections Based on a Microfluidic Nanodroplet Sample Preparation Platform.

Conventional proteomic approaches measure the averaged signal from mixed cell populations or bulk tissues, leading to the dilution of signals arising from subpopulations of cells that might serve as important biomarkers. Recent developments in bottom-up proteomics have enabled spatial mapping of cellular heterogeneity in tissue microenvironments. However, bottom-up proteomics cannot unambiguously define and quantify proteoforms, which are intact (i.e., functional) forms of proteins capturing genetic variations, alternatively spliced transcripts and posttranslational modifications. Herein, we described a spatially resolved top-down proteomics (TDP) platform for proteoform identification and quantitation directly from tissue sections. The spatial TDP platform consisted of a nanodroplet processing in one pot for trace samples-based sample preparation system and an laser capture microdissection-based cell isolation system. We improved the nanodroplet processing in one pot for trace samples sample preparation by adding benzonase in the extraction buffer to enhance the coverage of nucleus proteins. Using ∼200 cultured cells as test samples, this approach increased total proteoform identifications from 493 to 700; with newly identified proteoforms primarily corresponding to nuclear proteins. To demonstrate the spatial TDP platform in tissue samples, we analyzed laser capture microdissection-isolated tissue voxels from rat brain cortex and hypothalamus regions. We quantified 509 proteoforms within the union of top-down mass spectrometry-based proteoform identification and characterization and TDPortal identifications to match with features from protein mass extractor. Several proteoforms corresponding to the same gene exhibited mixed abundance profiles between two tissue regions, suggesting potential posttranslational modification-specific spatial distributions. The spatial TDP workflow has prospects for biomarker discovery at proteoform level from small tissue sections.

IsoForma: An R Package for Quantifying and Visualizing Positional Isomers in Top-Down LC-MS/MS Data.

Proteoforms, the different forms of a protein with sequence variations including post-translational modifications (PTMs), execute vital functions in biological systems, such as cell signaling and epigenetic regulation. Advances in top-down mass spectrometry (MS) technology have permitted the direct characterization of intact proteoforms and their exact number of modification sites, allowing for the relative quantification of positional isomers (PI). Protein positional isomers refer to a set of proteoforms with identical total mass and set of modifications, but varying PTM site combinations. The relative abundance of PI can be estimated by matching proteoform-specific fragment ions to top-down tandem MS (MS2) data to localize and quantify modifications. However, the current approaches heavily rely on manual annotation. Here, we present IsoForma, an open-source R package for the relative quantification of PI within a single tool. Benchmarking IsoForma's performance against two existing workflows produced comparable results and improvements in speed. Overall, IsoForma provides a streamlined process for quantifying PI, reduces the analysis time, and offers an essential framework for developing customized proteoform analysis workflows. The software is open source and available at https://github.com/EMSL-Computing/isoforma-lib.

TopPICR: A Companion R Package for Top-Down Proteomics Data Analysis.

Top-down proteomics is the analysis of proteins in their intact form without proteolysis, thus preserving valuable information about post-translational modifications, isoforms, and proteolytic processing. However, it is still a developing field due to limitations in the instrumentation, difficulties with the interpretation of complex mass spectra, and a lack of well-established quantification approaches. TopPIC is one of the popular tools for proteoform identification. We extended its capabilities into label-free proteoform quantification by developing a companion R package (TopPICR). Key steps in the TopPICR pipeline include filtering identifications, inferring a minimal set of protein accessions explaining the observed sequences, aligning retention times, recalibrating measured masses, clustering features across data sets, and finally compiling feature intensities using the match-between-runs approach. The output of the pipeline is an MSnSet object which makes downstream data analysis seamlessly compatible with packages from the Bioconductor project. It also provides the capability for visualizing proteoforms within the context of the parent protein sequence. The functionality of TopPICR is demonstrated on top-down LC-MS/MS data sets of 10 human-in-mouse xenografts of luminal and basal breast tumor samples.

Comparing Top-Down Proteoform Identification: Deconvolution, PrSM Overlap, and PTM Detection.

Generating top-down tandem mass spectra (MS/MS) from complex mixtures of proteoforms benefits from improvements in fractionation, separation, fragmentation, and mass analysis. The algorithms to match MS/MS to sequences have undergone a parallel evolution, with both spectral alignment and match-counting approaches producing high-quality proteoform-spectrum matches (PrSMs). This study assesses state-of-the-art algorithms for top-down identification (ProSight PD, TopPIC, MSPathFinderT, and pTop) in their yield of PrSMs while controlling false discovery rate. We evaluated deconvolution engines (ThermoFisher Xtract, Bruker AutoMSn, Matrix Science Mascot Distiller, TopFD, and FLASHDeconv) in both ThermoFisher Orbitrap-class and Bruker maXis Q-TOF data (PXD033208) to produce consistent precursor charges and mass determinations. Finally, we sought post-translational modifications (PTMs) in proteoforms from bovine milk (PXD031744) and human ovarian tissue. Contemporary identification workflows produce excellent PrSM yields, although approximately half of all identified proteoforms from these four pipelines were specific to only one workflow. Deconvolution algorithms disagree on precursor masses and charges, contributing to identification variability. Detection of PTMs is inconsistent among algorithms. In bovine milk, 18% of PrSMs produced by pTop and TopMG were singly phosphorylated, but this percentage fell to 1% for one algorithm. Applying multiple search engines produces more comprehensive assessments of experiments. Top-down algorithms would benefit from greater interoperability.

Genetically Encoded Crosslinking Enables Identification of Multivalent Ubiquitin-Deubiquitylating Enzyme Interactions.

Ubiquitin (Ub) proteoforms control nearly every aspect of eukaryotic cell biology through their diversity. Inspired by the widely used Ub C-terminal electrophiles (Ub-E), here we report the identification of multivalent binding of Ub with deubiquitylating enzymes (Dubs) using genetic code expansion (GCE) and crosslinking mass spectrometry. While the Ub-Es only gather structural information with the S1 Dub sites, we demonstrate that GCE of Ub with p-benzoyl-L-phenylalanine enables identification of interaction modes beyond the S1 site with a panel of Dubs of both eukaryotic and prokaryotic origin. Collectively, this represents the next generation of Ub-based affinity probes with a unique ability to unravel Ub interaction landscapes beyond what is afforded by cysteine-based chemistries.

Initiation of fatty acid biosynthesis in Pseudomonas putida KT2440.

Deciphering the mechanisms of bacterial fatty acid biosynthesis is crucial for both the engineering of bacterial hosts to produce fatty acid-derived molecules and the development of new antibiotics. However, gaps in our understanding of the initiation of fatty acid biosynthesis remain. Here, we demonstrate that the industrially relevant microbe Pseudomonas putida KT2440 contains three distinct pathways to initiate fatty acid biosynthesis. The first two routes employ conventional ß-ketoacyl-ACP synthase III enzymes, FabH1 and FabH2, that accept short- and medium-chain-length acyl-CoAs, respectively. The third route utilizes a malonyl-ACP decarboxylase enzyme, MadB. A combination of exhaustive in vivo alanine-scanning mutagenesis, in vitro biochemical characterization, X-ray crystallography, and computational modeling elucidate the presumptive mechanism of malonyl-ACP decarboxylation via MadB. Given that functional homologs of MadB are widespread throughout domain Bacteria, this ubiquitous alternative fatty acid initiation pathway provides new opportunities to target a range of biotechnology and biomedical applications.

Analysis of a macrophage carbamylated proteome reveals a function in post-translational modification crosstalk.

BACKGROUND: Lysine carbamylation is a biomarker of rheumatoid arthritis and kidney diseases. However, its cellular function is understudied due to the lack of tools for systematic analysis of this post-translational modification (PTM). METHODS: We adapted a method to analyze carbamylated peptides by co-affinity purification with acetylated peptides based on the cross-reactivity of anti-acetyllysine antibodies. We also performed immobilized-metal affinity chromatography to enrich for phosphopeptides, which allowed us to obtain multi-PTM information from the same samples. RESULTS: By testing the pipeline with RAW 264.7 macrophages treated with bacterial lipopolysaccharide, 7,299, 8,923 and 47,637 acetylated, carbamylated, and phosphorylated peptides were identified, respectively. Our analysis showed that carbamylation occurs on proteins from a variety of functions on sites with similar as well as distinct motifs compared to acetylation. To investigate possible PTM crosstalk, we integrated the carbamylation data with acetylation and phosphorylation data, leading to the identification 1,183 proteins that were modified by all 3 PTMs. Among these proteins, 54 had all 3 PTMs regulated by lipopolysaccharide and were enriched in immune signaling pathways, and in particular, the ubiquitin-proteasome pathway. We found that carbamylation of linear diubiquitin blocks the activity of the anti-inflammatory deubiquitinase OTULIN. CONCLUSIONS: Overall, our data show that anti-acetyllysine antibodies can be used for effective enrichment of carbamylated peptides. Moreover, carbamylation may play a role in PTM crosstalk with acetylation and phosphorylation, and that it is involved in regulating ubiquitination in vitro. Video Abstract.

AI-Accelerated Design of Targeted Covalent Inhibitors for SARS-CoV-2.

Direct-acting antivirals for the treatment of the COVID-19 pandemic caused by the SARS-CoV-2 virus are needed to complement vaccination efforts. Given the ongoing emergence of new variants, automated experimentation, and active learning based fast workflows for antiviral lead discovery remain critical to our ability to address the pandemic's evolution in a timely manner. While several such pipelines have been introduced to discover candidates with noncovalent interactions with the main protease (Mpro), here we developed a closed-loop artificial intelligence pipeline to design electrophilic warhead-based covalent candidates. This work introduces a deep learning-assisted automated computational workflow to introduce linkers and an electrophilic "warhead" to design covalent candidates and incorporates cutting-edge experimental techniques for validation. Using this process, promising candidates in the library were screened, and several potential hits were identified and tested experimentally using native mass spectrometry and fluorescence resonance energy transfer (FRET)-based screening assays. We identified four chloroacetamide-based covalent inhibitors of Mpro with micromolar affinities (KI of 5.27 µM) using our pipeline. Experimentally resolved binding modes for each compound were determined using room-temperature X-ray crystallography, which is consistent with the predicted poses. The induced conformational changes based on molecular dynamics simulations further suggest that the dynamics may be an important factor to further improve selectivity, thereby effectively lowering KI and reducing toxicity. These results demonstrate the utility of our modular and data-driven approach for potent and selective covalent inhibitor discovery and provide a platform to apply it to other emerging targets.

Integrated data-driven and experimental approaches to accelerate lead optimization targeting SARS-CoV-2 main protease.

Identification of potential therapeutic candidates can be expedited by integrating computational modeling with domain aware machine learning (ML) models followed by experimental validation in an iterative manner. Generative deep learning models can generate thousands of new candidates, however, their physiochemical and biochemical properties are typically not fully optimized. Using our recently developed deep learning models and a scaffold as a starting point, we generated tens of thousands of compounds for SARS-CoV-2 Mpro that preserve the core scaffold. We utilized and implemented several computational tools such as structural alert and toxicity analysis, high throughput virtual screening, ML-based 3D quantitative structure-activity relationships, multi-parameter optimization, and graph neural networks on generated candidates to predict biological activity and binding affinity in advance. As a result of these combined computational endeavors, eight promising candidates were singled out and put through experimental testing using Native Mass Spectrometry and FRET-based functional assays. Two of the tested compounds with quinazoline-2-thiol and acetylpiperidine core moieties showed IC[Formula: see text] values in the low micromolar range: [Formula: see text] [Formula: see text]M and 3.41±0.0015 [Formula: see text]M, respectively. Molecular dynamics simulations further highlight that binding of these compounds results in allosteric modulations within the chain B and the interface domains of the Mpro. Our integrated approach provides a platform for data driven lead optimization with rapid characterization and experimental validation in a closed loop that could be applied to other potential protein targets.

Online Hydrophilic Interaction Chromatography (HILIC) Enhanced Top-Down Mass Spectrometry Characterization of the SARS-CoV-2 Spike Receptor-Binding Domain.

SARS-CoV-2 cellular infection is mediated by the heavily glycosylated spike protein. Recombinant versions of the spike protein and the receptor-binding domain (RBD) are necessary for seropositivity assays and can potentially serve as vaccines against viral infection. RBD plays key roles in the spike protein's structure and function, and thus, comprehensive characterization of recombinant RBD is critically important for biopharmaceutical applications. Liquid chromatography coupled to mass spectrometry has been widely used to characterize post-translational modifications in proteins, including glycosylation. Most studies of RBDs were performed at the proteolytic peptide (bottom-up proteomics) or released glycan level because of the technical challenges in resolving highly heterogeneous glycans at the intact protein level. Herein, we evaluated several online separation techniques: (1) C2 reverse-phase liquid chromatography (RPLC), (2) capillary zone electrophoresis (CZE), and (3) acrylamide-based monolithic hydrophilic interaction chromatography (HILIC) to separate intact recombinant RBDs with varying combinations of glycosylations (glycoforms) for top-down mass spectrometry (MS). Within the conditions we explored, the HILIC method was superior to RPLC and CZE at separating RBD glycoforms, which differ significantly in neutral glycan groups. In addition, our top-down analysis readily captured unexpected modifications (e.g., cysteinylation and N-terminal sequence variation) and low abundance, heavily glycosylated proteoforms that may be missed by using glycopeptide data alone. The HILIC top-down MS platform holds great potential in resolving heterogeneous glycoproteins for facile comparison of biosimilars in quality control applications.

Enhanced Spatial Mapping of Histone Proteoforms in Human Kidney Through MALDI-MSI by High-Field UHMR-Orbitrap Detection.

Core histones including H2A, H2B, H3, and H4 are key modulators of cellular repair, transcription, and replication within eukaryotic cells, playing vital roles in the pathogenesis of disease and cellular responses to environmental stimuli. Traditional mass spectrometry (MS)-based bottom-up and top-down proteomics allows for the comprehensive identification of proteins and of post-translational modification (PTM) harboring proteoforms. However, these methodologies have difficulties preserving near-cellular spatial distributions because they typically require laser capture microdissection (LCM) and advanced sample preparation techniques. Herein, we coupled a matrix-assisted laser desorption/ionization (MALDI) source with a Thermo Scientific Q Exactive HF Orbitrap MS upgraded with ultrahigh mass range (UHMR) boards for the first demonstration of complementary high-resolution accurate mass (HR/AM) measurements of proteoforms up to 16.5 kDa directly from tissues using this benchtop mass spectrometer. The platform achieved isotopic resolution throughout the detected mass range, providing confident assignments of proteoforms with low ppm mass error and a considerable increase in duty cycle over other Fourier transform mass analyzers. Proteoform mapping of core histones was demonstrated on sections of human kidney at near-cellular spatial resolution, with several key distributions of histone and other proteoforms noted within both healthy biopsy and a section from a renal cell carcinoma (RCC) containing nephrectomy. The use of MALDI-MS imaging (MSI) for proteoform mapping demonstrates several steps toward high-throughput accurate identification of proteoforms and provides a new tool for mapping biomolecule distributions throughout tissue sections in extended mass ranges.

AutoCCS: automated collision cross-section calculation software for ion mobility spectrometry-mass spectrometry.

MOTIVATION: Ion mobility spectrometry (IMS) separations are increasingly used in conjunction with mass spectrometry (MS) for separation and characterization of ionized molecular species. Information obtained from IMS measurements includes the ion's collision cross section (CCS), which reflects its size and structure and constitutes a descriptor for distinguishing similar species in mixtures that cannot be separated using conventional approaches. Incorporating CCS into MS-based workflows can improve the specificity and confidence of molecular identification. At present, there is no automated, open-source pipeline for determining CCS of analyte ions in both targeted and untargeted fashion, and intensive user-assisted processing with vendor software and manual evaluation is often required. RESULTS: We present AutoCCS, an open-source software to rapidly determine CCS values from IMS-MS measurements. We conducted various IMS experiments in different formats to demonstrate the flexibility of AutoCCS for automated CCS calculation: (i) stepped-field methods for drift tube-based IMS (DTIMS), (ii) single-field methods for DTIMS (supporting two calibration methods: a standard and a new enhanced method) and (iii) linear calibration for Bruker timsTOF and non-linear calibration methods for traveling wave based-IMS in Waters Synapt and Structures for Lossless Ion Manipulations. We demonstrated that AutoCCS offers an accurate and reproducible determination of CCS for both standard and unknown analyte ions in various IMS-MS platforms, IMS-field methods, ionization modes and collision gases, without requiring manual processing. AVAILABILITY AND IMPLEMENTATION: https://github.com/PNNL-Comp-Mass-Spec/AutoCCS. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online. Demo datasets are publicly available at MassIVE (Dataset ID: MSV000085979).

Relative interfacial cleavage energetics of protein complexes revealed by surface collisions.

To fulfill their biological functions, proteins must interact with their specific binding partners and often function as large assemblies composed of multiple proteins or proteins plus other biomolecules. Structural characterization of these complexes, including identification of all binding partners, their relative binding affinities, and complex topology, is integral for understanding function. Understanding how proteins assemble and how subunits in a complex interact is a cornerstone of structural biology. Here we report a native mass spectrometry (MS)-based method to characterize subunit interactions in globular protein complexes. We demonstrate that dissociation of protein complexes by surface collisions, at the lower end of the typical surface-induced dissociation (SID) collision energy range, consistently cleaves the weakest protein:protein interfaces, producing products that are reflective of the known structure. We present here combined results for multiple complexes as a training set, two validation cases, and four computational models. We show that SID appearance energies can be predicted from structures via a computationally derived expression containing three terms (number of residues in a given interface, unsatisfied hydrogen bonds, and a rigidity factor).

PSpecteR: A User-Friendly and Interactive Application for Visualizing Top-Down and Bottom-Up Proteomics Data in R.

Visual examination of mass spectrometry data is necessary to assess data quality and to facilitate data exploration. Graphics provide the means to evaluate spectral properties, test alternative peptide/protein sequence matches, prepare annotated spectra for publication, and fine-tune parameters during wet lab procedures. Visual inspection of LC-MS data is constrained by proteomics visualization software designed for particular workflows or vendor-specific tools without open-source code. We built PSpecteR, an open-source and interactive R Shiny web application for visualization of LC-MS data, with support for several steps of proteomics data processing, including reading various mass spectrometry files, running open-source database search engines, labeling spectra with fragmentation patterns, testing post-translational modifications, plotting where identified fragments map to reference sequences, and visualizing algorithmic output and metadata. All figures, tables, and spectra are exportable within one easy-to-use graphical user interface. Our current software provides a flexible and modern R framework to support fast implementation of additional features. The open-source code is readily available (https://github.com/EMSL-Computing/PSpecteR), and a PSpecteR Docker container (https://hub.docker.com/r/emslcomputing/pspecter) is available for easy local installation.

Enhancing Top-Down Proteomics of Brain Tissue with FAIMS.

Proteomic investigations of Alzheimer's and Parkinson's disease have provided valuable insights into neurodegenerative disorders. Thus far, these investigations have largely been restricted to bottom-up approaches, hindering the degree to which one can characterize a protein's "intact" state. Top-down proteomics (TDP) overcomes this limitation; however, it is typically limited to observing only the most abundant proteoforms and of a relatively small size. Therefore, fractionation techniques are commonly used to reduce sample complexity. Here, we investigate gas-phase fractionation through high-field asymmetric waveform ion mobility spectrometry (FAIMS) within TDP. Utilizing a high complexity sample derived from Alzheimer's disease (AD) brain tissue, we describe how the addition of FAIMS to TDP can robustly improve the depth of proteome coverage. For example, implementation of FAIMS with external compensation voltage (CV) stepping at -50, -40, and -30 CV could more than double the mean number of non-redundant proteoforms, genes, and proteome sequence coverage compared to without FAIMS. We also found that FAIMS can influence the transmission of proteoforms and their charge envelopes based on their size. Importantly, FAIMS enabled the identification of intact amyloid beta (Aß) proteoforms, including the aggregation-prone Aß1-42 variant which is strongly linked to AD. Raw data and associated files have been deposited to the ProteomeXchange Consortium via the MassIVE data repository with data set identifier PXD023607.

MicroPOTS Analysis of Barrett's Esophageal Cell Line Models Identifies Proteomic Changes after Physiologic and Radiation Stress.

Moving from macroscale preparative systems in proteomics to micro- and nanotechnologies offers researchers the ability to deeply profile smaller numbers of cells that are more likely to be encountered in clinical settings. Herein a recently developed microscale proteomic method, microdroplet processing in one pot for trace samples (microPOTS), was employed to identify proteomic changes in ∼200 Barrett's esophageal cells following physiologic and radiation stress exposure. From this small population of cells, microPOTS confidently identified >1500 protein groups, and achieved a high reproducibility with a Pearson's correlation coefficient value of R > 0.9 and over 50% protein overlap from replicates. A Barrett's cell line model treated with either lithocholic acid (LCA) or X-ray had 21 (e.g., ASNS, RALY, FAM120A, UBE2M, IDH1, ESD) and 32 (e.g., GLUL, CALU, SH3BGRL3, S100A9, FKBP3, AGR2) overexpressed proteins, respectively, compared to the untreated set. These results demonstrate the ability of microPOTS to routinely identify and quantify differentially expressed proteins from limited numbers of cells.

Author Correction: Informed-Proteomics: open-source software package for top-down proteomics.

In the version of this article initially published, the authors erroneously reported the search mode that was used for ProSightPC 3.0 in the Online Methods and in Supplementary Table 3.

De novo sequencing and native mass spectrometry revealed hetero-association of dirigent protein homologs and potential interacting proteins in Forsythia × intermedia.

The discovery of dirigent proteins (DPs) and their functions in plant phenol biochemistry was made over two decades ago with Forsythia × intermedia. Stereo-selective, DP-guided, monolignol-derived radical coupling in vitro was then reported to afford the optically active lignan, (+)-pinoresinol from coniferyl alcohol, provided one-electron oxidase/oxidant capacity was present. It later became evident that DPs have several distinct sub-families, presumably with different functions. Some known DPs require other essential enzymes/proteins (e.g. oxidases) for their functions. However, the lack of a fully sequenced genome for Forsythia × intermedia made it difficult to profile other components co-purified with the (+)-pinoresinol forming DP. Herein, we used an integrated bottom-up, top-down, and native mass spectrometry (MS) approach to de novo sequence the extracted proteins via adaptation of our initial report of DP solubilization and purification. Using publicly available transcriptome and genomic data from closely related species, we identified 14 proteins that were putatively associated with either DP function or the cell wall. Although their co-occurrence after extraction and chromatographic separation is suggestive for potential protein-protein interactions, none were found to form stable protein complexes with DPs in native MS under the specific experimental conditions we have explored. Interestingly, two new DP homologs were found and they formed hetero-trimers. Molecular dynamics simulations suggested that similar hetero-trimers were possible between Arabidopsis DP homologs with comparable sequence similarities. Nevertheless, our integrated mass spectrometry method development helped prepare for future investigations directed to the discovery of novel proteins and protein-protein interactions. These advantages can be highly beneficial for plant and microbial research where fully sequenced genomes may not be readily available.

Top-down mass spectrometry of histone modifications in sorghum reveals potential epigenetic markers for drought acclimation.

Sorghum [Sorghum bicolor (L.) Moench] is an important cereal crop noted for its ability to survive water-limiting conditions. Herein, we present an analytical workflow to explore the changes in histone modifications through plant developmental stages and two drought stresses in two sorghum genotypes that differ in their response to drought. Top-down mass spectrometry (MS) is an ideal method to profile histone modifications and distinguish closely related histone proteoforms. We analyzed leaves of 48 plants and identified 26 unique histone proteins and 677 unique histone proteoforms (124 full-length and 553 truncated proteoforms). We detected trimethylation on nearly all H2B N-termini where acetylation is commonly expected. In addition, an unexpected modification on H2A histones was assigned to N-pyruvic acid 2-iminylation based on its unique neutral loss of CO2. Interestingly, some of the truncated histones, in particular H4 and H3.2, showed significant changes that correlated with the growth and water conditions. The histone proteoforms could serve as targets in search of chromatin modifiers and ultimately have important ramifications in future attempts of studying plant epigenetic reprogramming under stress.