Defining the commonalities between post-transcriptional and post-translational modification communities.

Post-transcriptional modifications of RNA (PRMs) and post-translational modifications of proteins (PTMs) are important regulatory mechanisms in biological processes and have many commonalities. However, the integration of these research areas is lacking. A recent discussion identified the priorities, areas of emphasis, and necessary technologies to advance and integrate these areas of study.

Real-Time Spectral Library Matching for Sample Multiplexed Quantitative Proteomics.

Sample multiplexed quantitative proteomics assays have proved to be a highly versatile means to assay molecular phenotypes. Yet, stochastic precursor selection and precursor coisolation can dramatically reduce the efficiency of data acquisition and quantitative accuracy. To address this, intelligent data acquisition (IDA) strategies have recently been developed to improve instrument efficiency and quantitative accuracy for both discovery and targeted methods. Toward this end, we sought to develop and implement a new real-time spectral library searching (RTLS) workflow that could enable intelligent scan triggering and peak selection within milliseconds of scan acquisition. To ensure ease of use and general applicability, we built an application to read in diverse spectral libraries and file types from both empirical and predicted spectral libraries. We demonstrate that RTLS methods enable improved quantitation of multiplexed samples, particularly with consideration for quantitation from chimeric fragment spectra. We used RTLS to profile proteome responses to small molecule perturbations and were able to quantify up to 15% more significantly regulated proteins in half the gradient time compared to traditional methods. Taken together, the development of RTLS expands the IDA toolbox to improve instrument efficiency and quantitative accuracy for sample multiplexed analyses.

Identification of MPK4 interacting proteins in guard cells.

Plants as sessile organisms are challenged by numerous biotic and abiotic stresses. Stomatal guard cells on the leaf surface are at the frontline of biotic and abiotic stress responses. Mitogen-activated protein kinase 4 (MPK4) has higher expression levels in guard cells than in mesophyll cells. The specific functions of MPK4 in guard cells are unknown. In this study, when MPK4 was overexpressed in Arabidopsis, bacterial entry of Pseudomonas syringae (Pst) into the plants was significantly decreased. The MPK4 overexpression plants had a similar trend of stomatal movement as wild-type Col-0, but had a smaller stomatal aperture than the Col-0, highlighting MPK4 plays a role in stomatal immune response. This function of the MPK4 requires its kinase activity because the MPK4 kinase-dead mutant did not have a significant difference in stomatal aperture compared to the Col-0. To understand MPK4 functions in guard cells, we investigated MPK4-associated protein complexes in guard cells using affinity purification mass spectrometry. A total of 145 proteins were identified to be in the MPK4-complex. Ten potential MPK4-interacting proteins were cloned and tested for physical interactions with the MPK4 using a yeast two hybrid (Y2H) system. Four proteins were newly identified to interact directly with the MPK4. SIGNIFICANCE: MPK4 is highly abundant in stomatal guard cells, but its specific functions in guard cells are largely unknown. Through a bacterial entry assay of MPK4 overexpression plants, we found that MPK4 may play an important role in stomatal immune response. To understand the molecular mechanisms underlying the MPK4 functions in guard cells, we characterized the MPK4-associated protein complex in guard cells. Many of the 145 identified proteins were involved in plant immunity and development. Four of the proteins were newly identified to interact directly with the MPK4. This work has provided additional evidence for the MPK4 function as a positive regulator for stomatal immunity. The guard cell MPK4 protein complex and the four new interacting proteins were revealed. Whether MPK4 directly phosphorylates these interacting proteins deserves further investigation. These newly discovered proteins have chartered exciting directions toward understanding new functions of the MPK4 kinase.

Profiling Glycosylphosphatidylinositol (GPI)-Interacting Proteins in the Cell Membrane Using a Bifunctional GPI Analogue as the Probe.

Glycosylphosphatidylinositol (GPI) anchorage of cell surface proteins to the membrane is biologically important and ubiquitous in eukaryotes. However, GPIs do not contain long enough lipids to span the entire membrane bilayer. To transduce binding signals, GPIs must interact with other membrane components, but such interactions are difficult to define. Here, a new method was developed to explore GPI-interacting membrane proteins in live cell with a bifunctional analogue of the glucosaminylphosphatidylinositol motif conserved in all GPIs as a probe. This probe contained a diazirine functionality in the lipid and an alkynyl group on the glucosamine residue to respectively facilitate the cross-linkage of GPI-binding membrane proteins with the probe upon photoactivation and then the installation of biotin to the cross-linked proteins via a click reaction for affinity-based protein isolation and analysis. Profiling the proteins pulled down from the Hela cells revealed 94 unique and 18 overrepresented proteins compared to the control, and most of them are membrane proteins and many are GPI-related. The results have proved not only the concept of using the new bifunctional GPI probe to investigate GPI-binding membrane proteins but also the important role of inositol in the biological functions of GPI anchors and GPI-anchored proteins.

Identification of MPK4 kinase interactome using TurboID proximity labeling proteomics in Arabidopsis thaliana.

TurboID is a new and efficient proximity labeling system that was first developed in living mammalian cells. TurboID is a modified bacterial biotin ligase that can be fused to a bait protein, which can then modify proximal interacting proteins with biotin. Prey proteins subsequently labeled with biotin tags will be pulled down with streptavidin-coated beads and identified by mass spectrometry-based proteomics. TurboID has been recently applied to living plant cells and provided promising results in identification of interacting proteins. Mitogen-activated protein kinase 4 (MPK4) is important for plant growth, development, and defense; however, the molecular mechanisms underlying the range of MPK4 functions are not completely known. Here we use modern proteomics together with the TurboID in a proof-of-concept study to profile the MPK4 interactome and uncover the functions of MPK4 in plant signaling cascades.

Advances in Plant Metabolomics and Its Applications in Stress and Single-Cell Biology.

In the past two decades, the post-genomic era envisaged high-throughput technologies, resulting in more species with available genome sequences. In-depth multi-omics approaches have evolved to integrate cellular processes at various levels into a systems biology knowledge base. Metabolomics plays a crucial role in molecular networking to bridge the gaps between genotypes and phenotypes. However, the greater complexity of metabolites with diverse chemical and physical properties has limited the advances in plant metabolomics. For several years, applications of liquid/gas chromatography (LC/GC)-mass spectrometry (MS) and nuclear magnetic resonance (NMR) have been constantly developed. Recently, ion mobility spectrometry (IMS)-MS has shown utility in resolving isomeric and isobaric metabolites. Both MS and NMR combined metabolomics significantly increased the identification and quantification of metabolites in an untargeted and targeted manner. Thus, hyphenated metabolomics tools will narrow the gap between the number of metabolite features and the identified metabolites. Metabolites change in response to environmental conditions, including biotic and abiotic stress factors. The spatial distribution of metabolites across different organs, tissues, cells and cellular compartments is a trending research area in metabolomics. Herein, we review recent technological advancements in metabolomics and their applications in understanding plant stress biology and different levels of spatial organization. In addition, we discuss the opportunities and challenges in multiple stress interactions, multi-omics, and single-cell metabolomics.

Mitogen-Activated Protein Kinase 4-Regulated Metabolic Networks.

Mitogen-activated protein kinase 4 (MPK4) was first identified as a negative regulator of systemic acquired resistance. It is also an important kinase involved in many other biological processes in plants, including cytokinesis, reproduction, and photosynthesis. Arabidopsis thaliana mpk4 mutant is dwarf and sterile. Previous omics studies including genomics, transcriptomics, and proteomics have revealed new functions of MPK4 in different biological processes. However, due to challenges in metabolomics, no study has touched upon the metabolomic profiles of the mpk4 mutant. What metabolites and metabolic pathways are potentially regulated by MPK4 are not known. Metabolites are crucial components of plants, and they play important roles in plant growth and development, signaling, and defense. Here we used targeted and untargeted metabolomics to profile metabolites in the wild type and the mpk4 mutant. We found that in addition to the jasmonic acid and salicylic acid pathways, MPK4 is involved in polyamine synthesis and photosynthesis. In addition, we also conducted label-free proteomics of the two genotypes. The integration of metabolomics and proteomics data allows for an insight into the metabolomic networks that are potentially regulated by MPK4.

Proteomics data of SNF1-related protein kinase 2.4 interacting proteins revealed by immunoprecipitation-mass spectrometry.

Identification of kinase substrates is a prerequisite for elucidating the mechanism by which a kinase transduces internal or external stimuli to cellular responses. Conventional methods to profile this type of protein-protein interaction typically deal with one kinase-substrate pair at a time. Mass spectrometry-based proteomics, on the other hand, can determine putative kinase-substrate pairs at a large-scale in an unbiased manner. In this study, we identified the interacting partners of SNF1-related protein kinase 2.4 (SnRK2.4) via immunoprecipitation coupled with mass spectrometry. Proteins from stable transgenic Arabidopsis plants overexpressing a FLAG-tagged SnRK2.4 (cloned from Brassica napus) were pulled down using an anti-FLAG antibody. The protein components were then identified by mass spectrometry. In parallel, proteins from wild type plants were also analyzed, providing a list of nonspecific binding proteins that were further removed from the candidate SnRK2.4-interacting protein list. Our data identified over 30 putative SnRK2.4 interacting partners, which included many key players in stress responses, transport, and cellular metabolic processes.

Association between childhood trauma and depression: A moderated mediation analysis among normative Chinese college students.

BACKGROUND: Childhood trauma is a critical risk factor for depression. Many studies have investigated the pathway between childhood trauma and depression, especially the mediating or moderating effects of neuroticism or resilience, but the results were inconsistent and there was no full model of these interactive factors. In addition, high prevalence of depression existed in normative college students, and few studies focused on their pathway between childhood trauma and depressive scores. Therefore, this study intended to examine the relationships among childhood trauma, resilience, neuroticism and depressive scores in normative college students. METHODS: Normative college students (n = 404) aged 18-22 years were recruited as participants from universities in Guangzhou in 2019. The participants were asked to complete four self-report questionnaires, including the Childhood Trauma Questionnaire-Short Form (CTQ-SF), Conner-Davidson Resilience Scale (CD-RISC), NEO-Five Factor Inventory (NEO-FFI) and the Beck Depression Inventory-II (BDI-II). RESULTS: Results revealed that the effect of childhood trauma on depressive scores in normative college students was mediated by neuroticism. In addition, resilience moderated the association between childhood trauma and neuroticism. CONCLUSIONS: This study helps to elucidate the mechanism that underlined the pathway between childhood trauma and depressive scores in normative college students. These findings may give indications of developing measures to strengthen resilience and lower neuroticism in normative college students with childhood traumatic experiences.

Proteomics and phosphoproteomics revealed molecular networks of stomatal immune responses.

MAIN CONCLUSION: Dynamic protein and phosphoprotein profiles uncovered the overall regulation of stomata movement against pathogen invasion and phosphorylation states of proteins involved in ABA, SA, calcium and ROS signaling, which may modulate the stomatal immune response. Stomatal openings represent a major route of pathogen entry into the plant, and plants have evolved mechanisms to regulate stomatal aperture as innate immune response against bacterial invasion. However, the mechanisms underlying stomatal immunity are not fully understood. Taking advantage of high-throughput liquid chromatography mass spectrometry (LC-MS), we performed label-free proteomic and phosphoproteomic analyses of enriched guard cells in response to a bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. In total, 495 proteins and 1229 phosphoproteins were identified as differentially regulated. These proteins are involved in a variety of signaling pathways, including abscisic acid and salicylic acid hormone signaling, calcium and reactive oxygen species signaling. We also showed that dynamic changes of phosphoprotein WRKY transcription factors may play a crucial role in regulating stomata movement in plant immunity. The identified proteins/phosphoproteins and the pathways form interactive molecular networks to regulate stomatal immunity. This study has provided new insights into the multifaceted mechanisms of stomatal immunity. The differential proteins and phosphoproteins are potential targets for engineering or breeding of crops for enhanced pathogen defense.

MPK4 Phosphorylation Dynamics and Interacting Proteins in Plant Immunity.

Arabidopsis MAP kinase 4 (MPK4) has been proposed to be a negative player in plant immunity, and it is also activated by pathogen-associated molecular patterns (PAMPs), such as flg22. The molecular mechanisms by which MPK4 is activated and regulates plant defense remain elusive. In this study, we investigated Arabidopsis defense against a bacterial pathogen Pseudomonas syringae pv tomato ( Pst) DC3000 when Brassica napus MPK4 ( BnMPK4) is overexpressed. We showed an increase in pathogen resistance and suppression of jasmonic acid (JA) signaling in the BnMPK4 overexpressing (OE) plants. We also showed that the OE plants have increased sensitivity to flg22-triggered reactive oxygen species (ROS) burst in guard cells, which resulted in enhanced stomatal closure compared to wild-type (WT). During flg22 activation, dynamic phosphorylation events within and outside of the conserved TEY activation loop were observed. To elucidate how BnMPK4 functions during the defense response, we used immunoprecipitation coupled with mass spectrometry (IP-MS) to identify BnMPK4 interacting proteins in the absence and presence of flg22. Quantitative proteomic analysis revealed a shift in the MPK4-associated protein network, providing insight into the molecular functions of MPK4 at the systems level.