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.

Redox regulation of a guard cell SNF1-related protein kinase in Brassica napus, an oilseed crop.

Kinase-mediated phosphorylation is a pivotal regulatory process in stomatal responses to stresses. Through a redox proteomics study, a sucrose non-fermenting 1-related protein kinase (SnRK2.4) was identified to be redox-regulated in Brassica napus guard cells upon abscisic acid treatment. There are six genes encoding SnRK2.4 paralogs in B. napus Here, we show that recombinant BnSnRK2.4-1C exhibited autophosphorylation activity and preferentially phosphorylated the N-terminal region of B. napus slow anion channel (BnSLAC1-NT) over generic substrates. The in vitro activity of BnSnRK2.4-1C requires the presence of manganese (Mn2+). Phosphorylation sites of autophosphorylated BnSnRK2.4-1C were mapped, including serine and threonine residues in the activation loop. In vitro BnSnRK2.4-1C autophosphorylation activity was inhibited by oxidants such as H2O2 and recovered by active thioredoxin isoforms, indicating redox regulation of BnSnRK2.4-1C. Thiol-specific isotope tagging followed by mass spectrometry analysis revealed specific cysteine residues responsive to oxidant treatments. The in vivo activity of BnSnRK2.4-1C is inhibited by 15 min of H2O2 treatment. Taken together, these data indicate that BnSnRK2.4-1C, an SnRK preferentially expressed in guard cells, is redox-regulated with potential roles in guard cell signal transduction.

Oxidation and phosphorylation of MAP kinase 4 cause protein aggregation.

Mitogen-activated protein kinase (MPK) cascades are highly conserved signaling pathways that respond to environmental cues. Arabidopsis MPK4 has been identified as a stress-responsive protein kinase. Here we demonstrate that Brassica napus MPK4 (BnMPK4) is activated by hydrogen peroxide (H2O2) and phytohormone abscisic acid (ABA). Transient expression of a constitutively active BnMPK4 causes H2O2 production and cell death in Nicotiana benthamiana leaves. However, little is known about how H2O2 contributes to the regulation of MPK4 kinase function. Biochemical analysis revealed that recombinant BnMPK4 autophosphorylates on both threonine and tyrosine residues in the activation loop. In the presence of H2O2, phosphorylation of BnMPK4 caused protein aggregation in vitro. The aggregation of BnMPK4 could be reversed to the monomeric form by reducing reagents. Point-mutation of cysteine codons indicated that cysteine 232 is involved in protein aggregation. Our results suggest that BnMPK4 is involved in reactive oxygen species (ROS) signaling and metabolism, and its aggregation may be modulated by redox.

Genome-wide identification and homeolog-specific expression analysis of the SnRK2 genes in Brassica napus guard cells.

Sucrose non-fermenting-1-related protein kinase 2 (SnRK2) proteins constitute a small plant-specific serine/threonine kinase family involved in abscisic acid (ABA) signaling and plant responses to biotic and abiotic stresses. Although SnRK2s have been well-studied in Arabidopsis thaliana, little is known about SnRK2s in Brassica napus. Here we identified 30 putative sequences encoding 10 SnRK2 proteins in the B. napus genome and the expression profiles of a subset of 14 SnRK2 genes in guard cells of B. napus. In agreement with its polyploid origin, B. napus maintains both homeologs from its diploid parents. The results of quantitative real-time PCR (qRT-PCR) and reanalysis of RNA-Seq data showed that certain BnSnRK2 genes were commonly expressed in leaf tissues in different varieties of B. napus. In particular, qRT-PCR results showed that 12 of the 14 BnSnRK2s responded to drought stress in leaves and in ABA-treated guard cells. Among them, BnSnRK2.4 and BnSnRK2.6 were of interest because of their robust responsiveness to ABA treatment and drought stress. Notably, BnSnRK2 genes exhibited up-regulation of different homeologs, particularly in response to abiotic stress. The homeolog expression bias in BnSnRK2 genes suggests that parental origin of genes might be responsible for efficient regulation of stress responses in polyploids. This work has laid a foundation for future functional characterization of the different BnSnKR2 homeologs in B. napus and its parents, especially their functions in guard cell signaling and stress responses.

Protein-protein interactions in plant mitogen-activated protein kinase cascades.

Mitogen-activated protein kinases (MAPKs) form tightly controlled signaling cascades that play essential roles in plant growth, development, and defense. However, the molecular mechanisms underlying MAPK cascades are still elusive, due largely to our poor understanding of how they relay the signals. Extensive effort has been devoted to characterization of MAPK-substrate interactions to illustrate phosphorylation-based signaling. The diverse MAPK substrates identified also shed light on how spatiotemporal-specific protein-protein interactions function in distinct MAPK cascade-mediated biological processes. This review surveys various technologies used for characterizing MAPK-substrate interactions and presents case studies of MPK4 and MPK6, highlighting the multiple functions of MAPKs. Mass spectrometry-based approaches in identifying MAPK-interacting proteins are emphasized due to their increasing utility and effectiveness. The potential for using MAPKs and their substrates in enhancing plant stress tolerance is also discussed.

Thiol-based redox proteins in abscisic acid and methyl jasmonate signaling in Brassica napus guard cells.

Reversibly oxidized cysteine sulfhydryl groups serve as redox sensors or targets of redox sensing that are important in various physiological processes. However, little is known about redox-sensitive proteins in guard cells and how they function in stomatal signaling. In this study, Brassica napus guard-cell proteins altered by redox in response to abscisic acid (ABA) or methyl jasmonate (MeJA) were identified by complementary proteomics approaches, saturation differential in-gel electrophoresis and isotope-coded affinity tagging. In total, 65 and 118 potential redox-responsive proteins were identified in ABA- and MeJA-treated guard cells, respectively. All the proteins contain at least one cysteine, and over half of them are predicted to form intra-molecular disulfide bonds. Most of the proteins fall into the functional groups of 'energy', 'stress and defense' and 'metabolism'. Based on the peptide sequences identified by mass spectrometry, 30 proteins were common to ABA- and MeJA-treated samples. A total of 44 cysteines were mapped in the identified proteins, and their levels of redox sensitivity were quantified. Two of the proteins, a sucrose non-fermenting 1-related protein kinase and an isopropylmalate dehydrogenase, were confirmed to be redox-regulated and involved in stomatal movement. This study creates an inventory of potential redox switches, and highlights a protein redox regulatory mechanism in ABA and MeJA signal transduction in guard cells.

Calcium-dependent protein kinases regulate polarized tip growth in pollen tubes.

Calcium signals are critical for the regulation of polarized growth in many eukaryotic cells, including pollen tubes and neurons. In plants, the regulatory pathways that code and decode Ca(2+) signals are poorly understood. In Arabidopsis thaliana, genetic evidence presented here indicates that pollen tube tip growth involves the redundant activity of two Ca(2+)-dependent protein kinases (CPKs), isoforms CPK17 and -34. Both isoforms appear to target to the plasma membrane, as shown by imaging of CPK17-yellow fluorescent protein (YFP) and CPK34-YFP in growing pollen tubes. Segregation analyses from two independent sets of T-DNA insertion mutants indicate that a double disruption of CPK17 and -34 results in an approximately 350-fold reduction in pollen transmission efficiency. The near sterile phenotype of homozygous double mutants could be rescued through pollen expression of a CPK34-YFP fusion. In contrast, a transgene rescue was blocked by mutations engineered to disrupt the Ca(2+)-activation mechanism of CPK34 (CPK34-YFP-E465A,E500A), providing in vivo evidence linking Ca(2+) activation to a biological function of a CPK. While double mutant pollen tubes displayed normal morphology, relative growth rates for the most rapidly growing tubes were reduced by more than three-fold compared with wild type. In addition, while most mutant tubes appeared to grow far enough to reach ovules, the vast majority (>90%) still failed to locate and fertilize ovules. Together, these results provide genetic evidence that CPKs are essential to pollen fitness, and support a mechanistic model in which CPK17 and -34 transduce Ca(2+) signals to increase the rate of pollen tube tip growth and facilitate a response to tropism cues.

Plant immune responses - from guard cells and local responses to systemic defense against bacterial pathogens.

When plants are infected by pathogens two distinct responses can occur, the early being a local response in the infected area, and later a systemic response in non-infected tissues. Closure of stomata has recently been found to be a local response to bacterial pathogens. Stomata closure is linked to both salicylic acid (SA), an essential hormone in local responses and systemic acquired resistance (SAR), and absisic acid (ABA) a key regulator of drought and other abiotic stresses. SAR reduces the effects of later infections. In this review we discuss recent research elucidating the role of guard cells in local and systemic immune responses, guard cell interactions with abiotic and hormone signals, as well as putative functions and interactions between long-distance SAR signals.

Characterization of thiol-based redox modifications of Brassica napusSNF1-related protein kinase 2.6-2C.

Sucrose nonfermenting 1-related protein kinase 2.6 (SnRK2.6), also known as Open Stomata 1 (OST1) in Arabidopsis thaliana, plays a pivotal role in abscisic acid (ABA)-mediated stomatal closure. Four SnRK2.6 paralogs were identified in the Brassica napus genome in our previous work. Here we studied one of the paralogs, BnSnRK2.6-2C, which was transcriptionally induced by ABA in guard cells. Recombinant BnSnRK2.6-2C exhibited autophosphorylation activity and its phosphorylation sites were mapped. The autophosphorylation activity was inhibited by S-nitrosoglutathione (GSNO) and by oxidized glutathione (GSSG), and the inhibition was reversed by reductants. Using monobromobimane (mBBr) labeling, we demonstrated a dose-dependent modification of BnSnRK2.6-2C by GSNO. Furthermore, mass spectrometry analysis revealed previously uncharacterized thiol-based modifications including glutathionylation and sulfonic acid formation. Of the six cysteine residues in BnSnRK2.6-2C, C159 was found to have different types of thiol modifications, suggesting its high redox sensitivity and versatility. In addition, mBBr labeling on tyrosine residues was identified. Collectively, these data provide detailed biochemical characterization of redox-induced modifications and changes of the BnSnRK2.6-2C activity.

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.

The CDPK superfamily of protein kinases.

The CDPK superfamily consists of six types of protein kinases, which differ in the regulatory domains they contain. CDPKs (calcium-dependent protein kinases or calmodulin-like domain protein kinases) are activated by the binding of calcium to their calmodulin-like regulatory domains. The carboxyl terminal domains of CRKs (CDPK-related kinases) have sequence similarity to the regulatory domains of CDPKs, but do not bind calcium. PPCKs (PEP carboxylase kinases) contain only a catalytic domain. PRKs (PPCK-related kinases) have a carboxyl-terminal domain that has no similarity to that of any other member of the superfamily. CCaMKs (calcium and calmodulin regulated kinases) bind both calcium ions and the calcium/calmodulin complex, whereas CaMKs (calmodulin-dependent protein kinases) bind the calcium/calmodulin complex, but not calcium. Phylogenetic trees constructed from amino acid sequences of catalytic or regulatory domains show that CDPKs and CRKs are closely related and might share a common ancestor. Plant CCaMKs and CaMK form a group more closely related to protozoan, than to plant, CDPKs. Intron analysis of the 42 CDPK, CRK, PPCK, and PRK genes from Arabidopsis supports the structure of the gene trees, the possibility that PPCKs/PRKs belong to the CDPK superfamily, and suggests that several introns have been added during evolution of the family.

Calcium-regulated protein kinases of plants.

The importance of calcium ions in coupling physiological responses to external and developmental signals in plants has been well documented. Recently, Plieth and Trewavas (Plant Physiology, 2002, 129: 786-796) have shown that gravistimulation, too, elicits changes in the concentration of cytoplasmic Ca2+ in Arabidopsis plants. Cytoplasmic calcium brings about responses by interacting with target proteins, many of which contain EF-hand calcium-binding motifs. In plants there are at least five classes of protein kinases, all of which are in the CDPK/SnRK family, that either contain EF-hands within their structure or interact with proteins that contain EF-hands. Calcium-dependent protein kinases (CDPKs) and calcium and calmodulin-activated protein kinases (CCaMKs) both contain EF hands in their C-terminal domains and are activated by the binding of calcium. SnRK3s (Group 3 of the SNF-1 related kinases) bind to proteins that contain three EF hands, and some are activated by calcium. Members of two other protein kinase classes, plant calmodulin-dependent protein kinase (CaMK) and CDPK-related kinase (CRK), bind to calmodulin, but it remains to be seen whether the activity of these enzymes is regulated by calcium/calmodulin. This paper will review what is known about the structure and the regulation of these protein kinases and address the question of why there is such a plethora of calcium-regulated kinases in plants.

Protein phosphorylation in stomatal movement.

As research progresses on how guard cells perceive and transduce environmental cues to regulate stomatal movement, plant biologists are discovering key roles of protein phosphorylation. Early research efforts focused on characterization of ion channels and transporters in guard cell hormonal signaling. Subsequent genetic studies identified mutants of kinases and phosphatases that are defective in regulating guard cell ion channel activities, and recently proteins regulated by phosphorylation have been identified. Here we review the essential role of protein phosphorylation in ABA-induced stomatal closure and in blue light-induced stomatal opening. We also highlight evidence for the cross-talk between different pathways, which is mediated by protein phosphorylation.

Calcium dependent protein kinase (CDPK) expression during fruit development in cultivated peanut (Arachis hypogaea) under Ca²âº-sufficient and -deficient growth regimens.

Adequate soil calcium (Ca²âº) levels are crucial for sustained reproductive development of peanut (Arachis hypogaea). A role for calcium dependent protein kinase was evaluated during peanut fruit development under sufficient and deficient soil Ca²âº conditions. Quantitative RT-PCR and protein gel blot analyses confirmed transcriptional upregulation of CDPK in seeds developing under inadequate soil Ca²âº regimen, as well as spatiotemporal regulation of CDPK expression during early mitotic growth and later during the storage phase of seed development. However, a consistent basal level of CDPK was present during similar developmental stages of pod tissue, irrespective of the soil Ca²âº status. Immunolocalization data showed CDPK decoration primarily in the outer most cell layers of the pericarp and around vascular bundles linked by lateral connections in developing pods, as well as the single vascular trace supplying nutrients to the developing seed. Finally, carbohydrate analyses and qRT-PCR data are provided for peanut genes encoding enzymes involved in sucrose cleavage (orthologs of Vicia faba, VfCWI1 and VfCWI2) and utilization (AhSuSy and AhSpS), and oleosin gene transcripts (AhOleo17.8 and AhOleo18.5) validating a role for CDPK in the establishment and maintenance of sink strength, and subsequent onset of storage product biosynthetic phase during seed maturation.

Calcium-dependent protein kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates.

The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca(2+)-dependent protein kinases (CPKs). While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16, and 34). Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with K(M) ∼70 µM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites). Of these, 74 (27%) were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies.

Advances in plant proteomics.

With the avalanche of genomic information and improvements in analytical technology, proteomics is becoming increasingly important for the study of many different aspects of plant functions. Since proteins serve as important components of major signaling and biochemical pathways, studies at protein levels are essential to reveal molecular mechanisms underlying plant growth, development, and interactions with the environment. The plant proteome is highly complex and dynamic. Although great strides need to be taken towards the ultimate goal of characterizing all the proteins in a proteome, current technologies have provided immense opportunities for high-throughput proteomic studies that have gone beyond simple protein identification to analyzing various functional aspects, such as quantification, PTM, subcellular localization, and protein-protein interactions. In this review of plant proteomics, advances in protein fractionation, separation, and MS will be outlined. Focus will be on recent development in functional analysis of plant proteins, which paves the way towards the comprehensive integration with transcriptomics, metabolomics, and other large scale "-omics" into systems biology.

Calcium-regulated phosphorylation of soybean serine acetyltransferase in response to oxidative stress.

Glycine max serine acetyltransferase 2;1 (GmSerat2;1) is a member of a family of enzymes that catalyze the first reaction in the biosynthesis of cysteine from serine. It was identified by interaction cloning as a protein that binds to calcium-dependent protein kinase. In vitro phosphorylation assays showed that GmSerat2;1, but not GmSerat2;1 mutants (S378A or S378D), were phosphorylated by soybean calcium-dependent protein kinase isoforms. Recombinant GmSerat2;1 was also phosphorylated by soybean cell extract in a Ca2+-dependent manner. Phosphorylation of recombinant GmSerat2;1 had no effect on its catalytic activity but rendered the enzyme insensitive to the feedback inhibition by cysteine. In transient expression analyses, fluorescently tagged GmSerat2;1 localized in the cytoplasm and with plastids. Phosphorylation state-specific antibodies showed that an increase in GmSerat2;1 phosphorylation occurred in vivo within 5 min of treatment of soybean cells with 0.5 mM hydrogen peroxide, whereas GmSerat2;1 protein synthesis was not significantly induced until 1 h after oxidant challenge. Internal Ca2+ was required in the induction of both GmSerat2;1 phosphorylation and synthesis. Treatment of cells with calcium antagonists showed that externally derived Ca2+ was important for retaining GmSerat2;1 at a basal level of phosphorylation but was not necessary for its hydrogen peroxide-induced synthesis. Protein phosphatase type 1, but not type 2A or alkaline phosphatase, dephosphorylated native GmSerat2;1 in vitro. These results support the hypothesis that GmSerat2;1 is regulated by calcium-dependent protein kinase phosphorylation in vivo and suggest that increased GmSerat2;1 synthesis and phosphorylation in response to active oxygen species could play a role in anti-oxidative stress response.

A phyloproteomic characterization of in vitro autophosphorylation in calcium-dependent protein kinases.

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.

A wound-responsive and phospholipid-regulated maize calcium-dependent protein kinase.

Using protein sequence data obtained from a calcium- and phospholipid-regulated protein kinase purified from maize (Zea mays), we isolated a cDNA encoding a calcium-dependent protein kinase (CDPK), which we designated ZmCPK11. The deduced amino acid sequence of ZmCPK11 includes the sequences of all the peptides obtained from the native protein. The ZmCPK11 sequence contains the kinase, autoregulatory, and calmodulin-like domains typical of CDPKs. Transcripts for ZmCPK11 were present in every tested organ of the plant, relatively high in seeds and seedlings and lower in stems, roots, and leaves. In leaves, kinase activity and ZmCPK11 mRNA accumulation were stimulated by wounding. The level of ZmCPK11 is also increased in noninjured neighboring leaves. The results suggest that the maize protein kinase is involved in a systemic response to wounding. Bacterially expressed glutathione S-transferase (GST)-ZmCPK11 was catalytically active in a calcium-dependent manner. Like the native enzyme, GST-ZmCPK11 was able to phosphorylate histone III-S and Syntide 2. Phosphorylation of histone was stimulated by phosphatidylserine, phosphatidylinositol, and phosphatidic acid, whereas phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, diolein, and cardiolipin did not increase the enzymatic activity. Autophosphorylation of GST-ZmCPK11 was stimulated by calcium and by phosphatidic acid and, to a lesser extent, by phosphatidylserine. Phosphatidylcholine did not affect autophosphorylation. These data unequivocally identify the maize phospholipid- and calcium-regulated protein kinase, which has protein kinase C-like activity, as a CDPK, and emphasize the potential that other CDPKs are regulated by phospholipids in addition to calcium.