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.

Proteomic characterization of MPK4 signaling network and putative substrates.

KEY MESSAGE: Combining genetic engineering of MPK4 activity and quantitative proteomics, we established an in planta system that enables rapid study of MPK4 signaling networks and potential substrate proteins. Mitogen activated protein kinase 4 (MPK4) is a multifunctional kinase that regulates various signaling events in plant defense, growth, light response and cytokinesis. The question of how a single protein modulates many distinct processes has spurred extensive research into the physiological outcomes resulting from genetic perturbation of MPK4. However, the mechanism by which MPK4 functions is still poorly understood due to limited data on the MPK4 networks including substrate proteins and downstream pathways. Here we introduce an experimental system that combines genetic engineering of kinase activity and quantitative proteomics to rapidly study the signaling networks of MPK4. First, we transiently expressed a constitutively active (MPK4CA) and an inactive (MPK4IN) version of a Brassica napus MPK4 (BnMPK4) in Nicotiana benthamiana leaves. Proteomics analysis revealed that BnMPK4 activation affects multiple pathways (e.g., metabolism, redox regulation, jasmonic acid biosynthesis and stress responses). Furthermore, BnMPK4 activation also increased protein phosphorylation in the phosphoproteome, from which putative MPK4 substrates were identified. Using protein kinase assay, we validated that a transcription factor TCP8-like (TCP8) and a PP2A regulatory subunit TAP46-like (TAP46) were indeed phosphorylated by BnMPK4. Taken together, we demonstrated the utility of proteomics and phosphoproteomics in elucidating kinase signaling networks and in identification of downstream substrates.

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.

Quantitative proteomics reveals a role of JAZ7 in plant defense response to Pseudomonas syringae DC3000.

Jasmonate ZIM-domain (JAZ) proteins are key transcriptional repressors regulating various biological processes. Although many studies have studied JAZ proteins by genetic and biochemical analyses, little is known about JAZ7-associated global protein networks and how JAZ7 contributes to bacterial pathogen defense. In this study, we aim to fill this knowledge gap by conducting unbiased large-scale quantitative proteomics using tandem mass tags (TMT). We compared the proteomes of a JAZ7 knock-out line, a JAZ7 overexpression line, as well as the wild type Arabidopsis plants in the presence and absence of Pseudomonas syringae DC3000 infection. Both pairwise comparison and multi-factor analysis of variance reveal that differential proteins are enriched in biological processes such as primary and secondary metabolism, redox regulation, and response to stress. The differential regulation in these pathways may account for the alterations in plant size, redox homeostasis and accumulation of glucosinolates. In addition, possible interplay between genotype and environment is suggested as the abundance of seven proteins is influenced by the interaction of the two factors. Collectively, we demonstrate a role of JAZ7 in pathogen defense and provide a list of proteins that are uniquely responsive to genetic disruption, pathogen infection, or the interaction between genotypes and environmental factors. SIGNIFICANCE: We report proteomic changes as a result of genetic perturbation of JAZ7, and the contribution of JAZ7 in plant immunity. Specifically, the similarity between the proteomes of a JAZ7 knockout mutant and the wild type plants confirmed the functional redundancy of JAZs. In contrast, JAZ7 overexpression plants were much different, and proteomic analysis of the JAZ7 overexpression plants under Pst DC3000 infection revealed that JAZ7 may regulate plant immunity via ROS modulation, energy balance and glucosinolate biosynthesis. Multiple variate analysis for this two-factor proteomics experiment suggests that protein abundance is determined by genotype, environment and the interaction between them.

Identification of MAPK Substrates Using Quantitative Phosphoproteomics.

Activation of mitogen-activated protein kinases (MAPKs) under diverse biotic and abiotic factors and identification of an array of downstream MAPK target proteins are hot topics in plant signal transduction. Through interactions with a plethora of substrate proteins, MAPK cascades regulate many physiological processes in the course of plant growth, development, and response to environmental factors. Identification and quantification of potential MAPK substrates are essential, but have been technically challenging. With the recent advancement in phosphoproteomics, here we describe a method that couples metal dioxide for phosphopeptide enrichment with tandem mass tags (TMT) mass spectrometry (MS) for large-scale MAPK substrate identification and quantification. We have applied this method to a transient expression system carrying a wild type (WT) and a constitutive active (CA) version of a MAPK. This combination of genetically engineered MAPKs and phosphoproteomics provides a high-throughput, unbiased analysis of MAPK-triggered phosphorylation changes on the proteome scale. Therefore, it is a robust method for identifying potential MAPK substrates and should be applicable in the study of other kinase cascades in plants as well as in other organisms.

Identification of thioredoxin targets in guard cell enriched epidermal peels using cysTMT proteomics.

Thioredoxins (Trx) play central roles in cellular redox regulation. Although hundreds of Trx targets have been identified using different approaches, the capture of targets in a quantitative and efficient manner is challenging. Here we report a high-throughput method using cysteine reactive tandem mass tag (cysTMT) labeling followed by liquid chromatography (LC)-mass spectrometry (MS) to screen for Trx targets. Compared to existing methods, this approach allows for i) three replicates of pairwise comparison in a single LC-MS run to reduce run-to-run variation; ii) efficient enrichment of cysteine-containing peptides that requires low protein input; and iii) accurate quantification of the cysteine redox status and localization of the Trx targeted cysteine residues. Application of this method in guard cell-enriched epidermal peels from Brassica napus revealed 80 Trx h targets involved in a broad range of processes, including photosynthesis, stress response, metabolism and cell signaling. The adaption of this protocol in other systems will greatly improve our understanding of the Trx function in regulating cellular redox homeostasis. BIOLOGICAL SIGNIFICANCE: Redox homeostasis is tightly regulated for proper cellular activities. Specific protein-protein interactions between redox active molecules such as thioredoxin (Trx) and target proteins constitute the basis for redox-regulated biological processes. The use of cysTMT quantitative proteomics for studying Trx reactions enabled identification of potential Trx targets that provide important insights into the redox regulation in guard cells, a specialized plant cell type responsible for sensing of environmental signals, gas exchange and plant productivity.