Jeffrey C. Anderson

Genetics and Phosphoproteomics Reveal a Protein Phosphorylation Network in the Abscisic Acid Signaling Pathway in Arabidopsis thaliana

A systems approach reveals how the SnRK2 family of kinases mediates abscisic acid signaling. SnRK2 Kinases in Plant Stress Signaling The SnRK2 subfamily of plant kinases is activated both by the hormone abscisic acid (ABA) and by dehydration stress in plants. Umezawa et al. analyzed the phosphoproteome and transcriptome of wild-type and SnRK2 triple-mutant plants exposed to ABA or dehydration. Motif analysis of the peptides that exhibited altered phosphorylation enabled the authors to identify both direct and indirect targets of SnRK2. The phosphoproteomic analysis indicated that SnRK2 primarily functioned in ABA signaling and regulated fewer targets in response to dehydration. Biochemical or genetic experiments confirmed the regulation of three of the SnRK2 targets in response to ABA: the mitogen-activated protein kinases AtMPK1 and AtMPK2, the transcription factor AREB1, and the previously uncharacterized protein SNS1. Abscisic acid (ABA) is a phytohormone that regulates diverse plant processes, including seed germination and the response to dehydration. In Arabidopsis thaliana, protein kinases of the SNF1-related protein kinase 2 (SnRK2) family are believed to transmit ABA- or dehydration-induced signals through phosphorylation of downstream substrates. By mass spectrometry, we identified proteins that were phosphorylated in Arabidopsis wild-type plants, but not in mutants lacking all three members of the SnRK2 family (srk2dei), treated with ABA or subjected to dehydration stress. The number of differentially phosphorylated peptides was greater in srk2dei plants treated with ABA than in the ones subjected to dehydration, suggesting that SnRK2 was mainly involved in ABA signaling rather than dehydration. We identified 35 peptides that were differentially phosphorylated in wild-type but not in srk2dei plants treated with ABA. Biochemical and genetic studies of candidate SnRK2-regulated phosphoproteins showed that SnRK2 promoted the ABA-induced activation of the mitogen-activated protein kinases AtMPK1 and AtMPK2; that SnRK2 mediated phosphorylation of Ser45 in a bZIP transcription factor, AREB1 (ABA-responsive element binding protein 1), and stimulated ABA-responsive gene expression; and that a previously unknown protein, SnRK2-substrate 1 (SNS1), was phosphorylated in vivo by ABA-activated SnRK2s. Reverse genetic analysis revealed that SNS1 inhibited ABA responses in Arabidopsis. Thus, by integrating genetics with phosphoproteomics, we identified multiple components of the ABA-responsive protein phosphorylation network.

MAP KINASE PHOSPHATASE1 and PROTEIN TYROSINE PHOSPHATASE1 Are Repressors of Salicylic Acid Synthesis and SNC1-Mediated Responses in Arabidopsis

Mitogen-activated protein (MAP) kinase phosphatases are important negative regulators of the levels and kinetics of MAP kinase activation that modulate cellular responses. The dual-specificity phosphatase MAP KINASE PHOSPHATASE1 (MKP1) was previously shown to regulate MAP KINASE6 (MPK6) activation levels and abiotic stress responses in Arabidopsis thaliana. Here, we report that the mkp1 null mutation in the Columbia (Col) accession results in growth defects and constitutive biotic defense responses, including elevated levels of salicylic acid, camalexin, PR gene expression, and resistance to the bacterial pathogen Pseudomonas syringae. PROTEIN TYROSINE PHOSPHATASE1 (PTP1) also interacts with MPK6, but the ptp1 null mutant shows no aberrant growth phenotype. However, the pronounced constitutive defense response of the mkp1 ptp1 double mutant reveals that MKP1 and PTP1 repress defense responses in a coordinated fashion. Moreover, mutations in MPK3 and MPK6 distinctly suppress mkp1 and mkp1 ptp1 phenotypes, indicating that MKP1 and PTP1 act as repressors of inappropriate MPK3/MPK6-dependent stress signaling. Finally, we provide evidence that the natural modifier of mkp1 in Col is largely the disease resistance gene homolog SUPPRESSOR OF npr1-1, CONSTITUTIVE 1 (SNC1) that is absent in the Wassilewskija accession. Our data thus indicate a major role of MKP1 and PTP1 in repressing salicylic acid biosynthesis in the autoimmune-like response caused by SNC1.

Arabidopsis MAP Kinase Phosphatase 1 (AtMKP1) negatively regulates MPK6-mediated PAMP responses and resistance against bacteria.

A primary component of plant defense is the detection of pathogen-associated molecular patterns (PAMPs) by plasma membrane-localized pathogen recognition receptors. PAMP perception results in rapid and transient activation of phosphorylation-dependent signaling pathways that lead to a wide array of defense-related responses, including extensive changes in gene expression. In Arabidopsis, several kinases, including the mitogen-activated protein kinases (MAPKs) MPK6 and MPK3, are rapidly activated after PAMP treatment, and are thought to positively regulate a wide array of defense-related responses. In contrast, negative regulation of PAMP responses by downstream phosphatases remains poorly understood. Here we report the identification of Arabidopsis MAP Kinase Phosphatase 1 (MKP1) as a negative regulator of diverse PAMP responses, including activation of MPK6 and MPK3, transient production of extracellular reactive oxygen species, accumulation of a subset of PAMP-regulated transcripts, and inhibition of seedling growth. In agreement with the enhanced PAMP response phenotypes observed in the mkp1 mutant, we found that mkp1 seedlings and adult plants are more resistant to the virulent bacterial pathogen Pseudomonas syringae pv. tomato (Pto) DC3000. Further genetic analysis revealed that MPK6, but not MPK3, is required for the mkp1-dependent increase in resistance to Pto and enhanced PAMP-induced growth inhibition observed in mkp1 seedlings. Together, our data support a role for MKP1 as a negative regulator of MPK6-mediated PAMP responses.

Decreased abundance of type III secretion system-inducing signals in Arabidopsis mkp1 enhances resistance against Pseudomonas syringae

Significance Pathogenic bacteria inject effector proteins into the host to suppress its defenses. However, bacteria produce the effector proteins and injection machinery only upon recognition of a potential host. Here we identified an Arabidopsis mutant, mapk phosphatase 1 (mkp1), with decreased levels of chemical signals recognized by the bacterium, thus making the plant more resistant by suppressing the ability of the pathogen, Pseudomonas syringae, to express and inject effector proteins. Reapplying these chemical signals not only eliminated resistance in the mkp1 mutant but also suppressed resistance in wild-type plants with a preinduced immune response. These results demonstrate an important layer in determining the biological outcome during host–pathogen interactions and may provide new targets for enhancing resistance against bacterial pathogens. Genes encoding the virulence-promoting type III secretion system (T3SS) in phytopathogenic bacteria are induced at the start of infection, indicating that recognition of signals from the host plant initiates this response. However, the precise nature of these signals and whether their concentrations can be altered to affect the biological outcome of host–pathogen interactions remain speculative. Here we use a metabolomic comparison of resistant and susceptible genotypes to identify plant-derived metabolites that induce T3SS genes in Pseudomonas syringae pv tomato DC3000 and report that mapk phosphatase 1 (mkp1), an Arabidopsis mutant that is more resistant to bacterial infection, produces decreased levels of these bioactive compounds. Consistent with these observations, T3SS effector expression and delivery by DC3000 was impaired when infecting the mkp1 mutant. The addition of bioactive metabolites fully restored T3SS effector delivery and suppressed the enhanced resistance in the mkp1 mutant. Pretreatment of plants with pathogen-associated molecular patterns (PAMPs) to induce PAMP-triggered immunity (PTI) also restricts T3SS effector delivery and enhances resistance by unknown mechanisms, and the addition of the bioactive metabolites similarly suppressed both aspects of PTI. Together, these results demonstrate that DC3000 perceives multiple signals derived from plants to initiate its T3SS and that the level of these host-derived signals impacts bacterial pathogenesis.

A simple and rapid technique for detecting protein phosphorylation using one-dimensional isoelectric focusing gels and immunoblot analysis.

SUMMARY We report a technique for detecting protein phosphorylation that involves isoelectric focusing in a vertical mini-gel format followed by immunoblot detection of the target protein. This method uses standard protein gel equipment, allows sensitive detection of protein phosphorylation when phosphospecific antibodies are not available, and provides a stoichiometric measure of phosphorylation. We demonstrate the application of this method for observing phosphorylation of an Arabidopsis thaliana protein in response to biotic stress.

Cell wall protection by the Candida albicans class I chitin synthases.

Highlights • Class I chitin synthases promote cell integrity during early polarized growth.• Class I enzymes show a unique and dynamic pattern of localization at septa.• Phosphorylation of Chs2 regulates the amount of protein localized to septation sites.• Cell wall stresses independently regulate the amount of Chs2 at specific sites.• Class I enzymes provide protection from cell wall stresses in early polarized growth.

Phosphorylation of Arabidopsis MAP Kinase Phosphatase 1 (MKP1) Is Required for PAMP Responses and Resistance against Bacteria

Phosphorylation of MKP1 is required for its function(s) in a subset of PAMP responses and for resistance to bacterial growth. Plants perceive potential pathogens via the recognition of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern recognition receptors, which initiates a series of intracellular responses that ultimately limit bacterial growth. PAMP responses include changes in intracellular protein phosphorylation, including the activation of mitogen-activated protein kinase (MAPK) cascades. MAP kinase phosphatases (MKPs), such as Arabidopsis (Arabidopsis thaliana) MKP1, are important negative regulators of MAPKs and play a crucial role in controlling the intensity and duration of MAPK activation during innate immune signaling. As such, the mkp1 mutant lacking MKP1 displays enhanced PAMP responses and resistance against the virulent bacterium Pseudomonas syringae pv tomato DC3000. Previous in vitro studies showed that MKP1 can be phosphorylated and activated by MPK6, suggesting that phosphorylation may be an important mechanism for regulating MKP1. We found that MKP1 was phosphorylated during PAMP elicitation and that phosphorylation stabilized the protein, resulting in protein accumulation after elicitation. MKP1 also can be stabilized by the proteasome inhibitor MG132, suggesting that MKP1 is constitutively degraded through the proteasome in the resting state. In addition, we investigated the role of MKP1 posttranslational regulation in plant defense by testing whether phenotypes of the mkp1 Arabidopsis mutant could be complemented by expressing phosphorylation site mutations of MKP1. The phosphorylation of MKP1 was found to be required for some, but not all, of MKP1’s functions in PAMP responses and defense against bacteria. Together, our results provide insight into the roles of phosphorylation in the regulation of MKP1 during PAMP signaling and resistance to bacteria.

Plasma membrane proteomics in the maize primary root growth zone: novel insights into root growth adaptation to water stress.

Previous work on maize (Zea mays L.) primary root growth under water stress showed that cell elongation is maintained in the apical region of the growth zone but progressively inhibited further from the apex. These responses involve spatially differential and coordinated regulation of osmotic adjustment, modification of cell wall extensibility, and other cellular growth processes that are required for root growth under water-stressed conditions. As the interface between the cytoplasm and the apoplast (including the cell wall), the plasma membrane likely plays critical roles in these responses. Using a simplified method for enrichment of plasma membrane proteins, the developmental distribution of plasma membrane proteins was analysed in the growth zone of well-watered and water-stressed maize primary roots. The results identified 432 proteins with differential abundances in well-watered and water-stressed roots. The majority of changes involved region-specific patterns of response, and the identities of the water stress-responsive proteins suggest involvement in diverse biological processes including modification of sugar and nutrient transport, ion homeostasis, lipid metabolism, and cell wall composition. Integration of the distinct, region-specific plasma membrane protein abundance patterns with results from previous physiological, transcriptomic and cell wall proteomic studies reveals novel insights into root growth adaptation to water stress.

Detection of Protein Phosphorylation and Charge Isoforms Using Vertical One-Dimensional Isoelectric Focusing Gels

During many biological responses, changes in protein modifications (e.g., phosphorylation) are often more critical than changes in protein abundance in determining the outcome of cellular responses. These important regulatory changes can alter a proteins location, activity, or binding partners. Monitoring modifications such as phosphorylation is often impeded, or even prevented, because of the need for specialized reagents and equipment that are expensive and/or time-consuming to produce. However, many protein modifications alter the isoelectric point (pI) of a protein. Therefore, we developed a denaturing, one-dimensional isoelectric focusing (IEF) procedure that separates proteins based on their pI to resolve different isoforms, allowing a relatively simple strategy for detecting changes in protein modifications. Although similar results can be achieved by two-dimensional gel electrophoresis, the method described here uses a multi-well SDS-PAGE format that allows many more samples to be assayed within a single gel, thereby greatly decreasing both the time and cost needed to assess modifications of a single protein in response many different treatment conditions. To increase the sensitivity of detection, we also optimized a procedure to transfer proteins from these gels to membranes for subsequent immunodetection. This combination of techniques provides the means of interrogating the number and stoichiometry of isoforms from total protein extracts without a priori knowledge of which modification may occur.

Protein Phosphorylation Network in Abscisic Acid Signaling

Abscisic acid (ABA) is an important phytohormone that regulates a diversity of plant processes including seed germination, abiotic stress responses, e.g., drought tolerance, or freezing tolerance. In Arabidopsis, protein kinases in subclass III of the SNF1-related protein kinase 2 (SnRK2) family function immediately downstream of ABA and are believed to transmit ABA-induced signals by phosphorylating downstream substrates. However, the identities of proteins phosphorylated by SnRK2s remain largely unknown. To increase our understanding of the regulation of ABA signaling pathways by SnRK2s, we used a mass spectrometry-based phosphoproteomics approach to screen for phosphorylated proteins in Arabidopsis wild-type and SnRK2-disrupted mutant (srk2dei) plants treated with ABA or dehydration stress. A total of 5,288 phosphopeptides were identified among all treatments, and comparative analysis between treatments revealed 269 phosphorylated or dephosphorylated peptides in response to ABA and/or dehydration. Also, 35 of the changes in phosphorylation occurred in WT but not srk2dei plants, suggesting that they are regulated either directly or indirectly by SnRK2s. Additional biochemical and genetic studies of several candidate SnRK2-regulated phosphoproteins identified multiple novel components of the ABA-responsive protein phosphorylation network. Our results demonstrate that comparative and mutant-oriented phosphoproteomics can be applied to gene discovery in plants.