[Show abstract][Hide abstract] ABSTRACT: Plants are protected from microbial infection by a robust immune system. Two of the earliest responses mediated by surface-localized immune receptors include an increase in cytosolic calcium (Ca(2+)) and a burst of apoplastic reactive oxygen species (ROS). The Arabidopsis plasma membrane-associated cytoplasmic kinase BIK1 is an immediate convergent substrate of multiple surface-localized immune receptors that is genetically required for the PAMP-induced Ca(2+) burst and directly regulates ROS production catalyzed by the NADPH oxidase RBOHD. We recently demonstrated that Arabidopsis plants maintain an optimal level of BIK1 through a process of continuous degradation regulated by the Ca(2+)-dependent protein kinase CPK28. cpk28 mutants accumulate more BIK1 protein and display enhanced immune signaling, while plants over-expressing CPK28 accumulate less BIK1 protein and display impaired immune signaling. Here, we show that CPK28 additionally contributes to the PAMP-induced Ca(2+) burst, supporting its role as a negative regulator of BIK1.
[Show abstract][Hide abstract] ABSTRACT: Plant perception of pathogen-associated molecular patterns (PAMPs) triggers a phosphorylation relay leading to PAMP-triggered immunity (PTI). Despite increasing knowledge of PTI signaling, how immune homeostasis is maintained remains largely unknown. Here we describe a forward-genetic screen to identify loci involved in PTI and characterize the Arabidopsis calcium-dependent protein kinase CPK28 as a negative regulator of immune signaling. Genetic analyses demonstrate that CPK28 attenuates PAMP-triggered immune responses and antibacterial immunity. CPK28 interacts with and phosphorylates the plasma-membrane-associated cytoplasmic kinase BIK1, an important convergent substrate of multiple pattern recognition receptor (PRR) complexes. We find that BIK1 is rate limiting in PTI signaling and that it is continuously turned over to maintain cellular homeostasis. We further show that CPK28 contributes to BIK1 turnover. Our results suggest a negative regulatory mechanism that continually buffers immune signaling by controlling the turnover of this key signaling kinase.
[Show abstract][Hide abstract] ABSTRACT: Ca2+ is a ubiquitous second messenger for cellular signalling in various stresses and developmental processes. Here, we summarize current developments in the roles of Ca2+ during plant immunity responses. We discuss the early perception events preceding and necessary for triggering cellular Ca2+ fluxes, the potential Ca2+-permeable channels, the decoding of Ca2+ signals predominantly via Ca2+-dependent phosphorylation events and transcriptional reprogramming. To highlight the complexity of the cellular signal network, we briefly touch on the interplay between Ca2+-dependent signalling and selected major signalling mechanisms – with special emphasis on reactive oxygen species at local and systemic levels.
New Phytologist 09/2014; 204(4). DOI:10.1111/nph.13031 · 7.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Calcium-dependent protein kinases (CDPKs) have been shown to play important roles in plant environmental stress signal transduction. We report on the identification of ZmCPK1 as a member of the maize (Zea mays) CDPK gene family involved in the regulation of the maize cold-stress response. Based on in silico analysis of the Zea mays cv. B73 genome, we identified that the maize CDPK gene family consists of 39 members. Two CDPK members were selected whose gene expression was either increased (Zmcpk1) or decreased (Zmcpk25) in response to cold exposure. Biochemical analysis demonstrated that ZmCPK1 displays calcium-independent protein kinase activity. The C-terminal calcium-binding domain of ZmCPK1 is sufficient to mediate calcium-independency of a previously calcium-dependent enzyme in chimeric ZmCPK25-CPK1 proteins. Furthermore, co-transfection of maize mesophyll protoplasts with active full-length ZmCPK1 suppressed the expression of a cold-induced marker gene, Zmerf3 (ZmCOI6.21). In accordance, heterologous overexpression of ZmCPK1 in Arabidopsis thaliana yielded plants with altered acclimation-induced frost tolerance. Our results identify ZmCPK1 as a negative regulator of cold stress signalling in maize.
[Show abstract][Hide abstract] ABSTRACT: Calcium-dependent protein kinases (CDPKs) are multifunctional proteins in which a calmodulin-like calcium-sensor and a protein kinase effector domain are combined in one molecule. Not surprisingly, CDPKs were primarily recognized as signaling mediators, which perceive rapid intracellular changes of Ca(2+) ion concentration, for example triggered by environmental stress cues, and relay them into specific phosphorylation events to induce further downstream stress responses. In the context of both, plant exposure to biotrophic pathogens-derived signals as well as plant attack by herbivores and wounding, CDPKs were shown to undergo rapid biochemical activation within seconds to minutes after stimulation and to induce local defence-responses including respective changes in gene expression patterns. In addition, CDPK function was correlated with the control of either salicylic acid-mediated or jasmonic acid-mediated phytohormone signaling pathways, mediating long term resistance to either biotrophic bacterial pathogens or herbivores also in distal parts of a plant. It has long been unclear how an individual enzyme can affect both rapid local as well as long-term distal immune responses. Here, we discuss recently raised topics from the field of CDPK research, in particular with a view on the identification of in vivo phosphorylation targets, which provide first mechanistic insights into the dual role of these enzymes: On the one hand as component of a self-activating circuit responsible for rapid plasma-membrane anchored cell-to-cell signal propagation from local to distal plant sites. On the other hand as nuclear-located regulators of transcription factor activity. Finally, we will highlight the dual function of calcium sensors in plasma-membrane/calcium-mediated signal propagation and in phytohormone signaling-dependent systemic resistance in immune responses to both, bacterial pathogens and herbivores.
Current opinion in plant biology 03/2014; 20C:1-10. DOI:10.1016/j.pbi.2014.03.002 · 7.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Reactive oxygen species (ROS) produced by NAD(P)H oxidases play a central role in plant stress response and development. To better understand the function of NAD(P)H oxidases in plant development we characterized the Arabidopsis thaliana NAD(P)H oxidases RBOHH and RBOHJ. Both proteins were specifically expressed in pollen and dynamically targeted to distinct and overlapping plasma membrane domains at the pollen tube tip. Functional loss of RBOHH and RBOHJ in homozygous double mutants resulted in reduced fertility. Analyses of pollen tube growth revealed remarkable differences in growth dynamics between Col-0 and rbohh-1 rbohj-2 pollen tubes. Growth rate oscillations of rbohh-1 rbohj-2 pollen tubes showed strong fluctuations in amplitude and frequency ultimately leading to pollen tube collapse. Prior to disintegration, rbohh-1 rbohj-2 pollen tubes exhibit high frequency growth oscillations, with significantly elevated growth rates, suggesting that an increase in the rate of cell wall exocytosis precedes pollen tube collapse. Time lapse imaging of exocytic dynamics revealed that NAD(P)H oxidases slow down pollen tube growth to coordinate the rate of cell expansion with the rate of exocytosis thereby dampening the amplitude of intrinsic growth oscillations. Using the Ca(2+) reporter Yellow Cameleon 3.6, we demonstrate that high amplitude growth rate oscillations in rbohh1- rbohj-2 pollen tubes are correlated with growth-dependent Ca(2+) bursts. Electrophysiological experiments involving double mutant pollen tubes and pharmacological treatments also showed that ROS can influence K(+) homeostasis. Our results indicate that, by limiting pollen tube growth, ROS produced by NAD(P)H oxidases modulate amplitude and frequency of pollen tube growth rate oscillations. This article is protected by copyright. All rights reserved.
The Plant Journal 02/2014; 78(1). DOI:10.1111/tpj.12452 · 5.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Calcium-dependent protein kinases (CDPKs) are multifunctional proteins in which a calmodulin-like calcium-sensor and a protein kinase effector domain are combined in one molecule. Not surprisingly, CDPKs were primarily recognized as signaling mediators, which perceive rapid intracellular changes of Ca2+ ion concentration, for example triggered by environmental stress cues, and relay them into specific phosphorylation events to induce further downstream stress responses. In the context of both, plant exposure to biotrophic pathogens-derived signals as well as plant attack by herbivores and wounding, CDPKs were shown to undergo rapid biochemical activation within seconds to minutes after stimulation and to induce local defence-responses including respective changes in gene expression patterns. In addition, CDPK function was correlated with the control of either salicylic acid-mediated or jasmonic acid-mediated phytohormone signaling pathways, mediating long term resistance to either biotrophic bacterial pathogens or herbivores also in distal parts of a plant. It has long been unclear how an individual enzyme can affect both rapid local as well as long-term distal immune responses. Here, we discuss recently raised topics from the field of CDPK research, in particular with a view on the identification of in vivo phosphorylation targets, which provide first mechanistic insights into the dual role of these enzymes: On the one hand as component of a self-activating circuit responsible for rapid plasma-membrane anchored cell-to-cell signal propagation from local to distal plant sites. On the other hand as nuclear-located regulators of transcription factor activity. Finally, we will highlight the dual function of calcium sensors in plasma-membrane/calcium-mediated signal propagation and in phytohormone signaling-dependent systemic resistance in immune responses to both, bacterial pathogens and herbivores.
[Show abstract][Hide abstract] ABSTRACT: Apical growth in pollen tubes (PTs) is associated with the presence of tip-focused ion gradients and fluxes, implying polar localization or regulation of the underlying transporters. The molecular identity and regulation of anion transporters in PTs is unknown. Here we report a negative gradient of cytosolic anion concentration focused on the tip, in negative correlation with the cytosolic Ca(2+) concentration. We hypothesized that a possible link between these two ions is based on the presence of Ca(2+)-dependent protein kinases (CPKs). We characterized anion channels and CPK transcripts in PTs and analyzed their localization. Yellow fluorescent protein (YFP) tagging of a homolog of SLOW ANION CHANNEL-ASSOCIATED1 (SLAH3:YFP) was widespread along PTs, but, in accordance with the anion efflux, CPK2/CPK20/CPK17/CPK34:YFP fluorescence was strictly localized at the tip plasma membrane. Expression of SLAH3 with either CPK2 or CPK20 (but not CPK17/CPK34) in Xenopus laevis oocytes elicited S-type anion channel currents. Interaction of SLAH3 with CPK2/CPK20 (but not CPK17/CPK34) was confirmed by Förster-resonance energy transfer fluorescence lifetime microscopy in Arabidopsis thaliana mesophyll protoplasts and bimolecular fluorescence complementation in living PTs. Compared with wild-type PTs, slah3-1 and slah3-2 as well as cpk2-1 cpk20-2 PTs had reduced anion currents. Double mutant cpk2-1 cpk20-2 and slah3-1 PTs had reduced extracellular anion fluxes at the tip. Our studies provide evidence for a Ca(2+)-dependent CPK2/CPK20 regulation of the anion channel SLAH3 to regulate PT growth.
The Plant Cell 11/2013; 25(11). DOI:10.1105/tpc.113.118463 · 9.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Calcium ions (Ca(2+)) have long been recognized as a major conserved second messenger in eukaryotic signal transduction. In plants, the multigene family of calcium-dependent protein kinases (CDPKs) encodes structurally conserved uni-molecular calcium-sensor/protein kinase effector proteins. This makes CDPKs ideal candidates for perceiving intracellular changes in Ca(2+) concentration and translating them into specific phosphorylation events to initiate further downstream signaling processes. In accordance, CDPKs were predominantly characterized in rapid plant abiotic stress and immune signaling responses, with only little focus laid on CDPK function in long term adaptive processes or plant development. The long awaited identification of CDPK in vivo/in planta targets as well as respective phosphorylation sites within these proteins not only elucidates CDPK function on a molecular and biochemical level. Even more, the resolution of kinase-specific phosphorylation patterns within a target protein provides mechanistic evidence suggesting a role of CDPKs as hubs in plant stress signaling and development.
[Show abstract][Hide abstract] ABSTRACT: In animals and plants, pathogen recognition triggers the local activation of intracellular signaling that is prerequisite for mounting systemic defenses in the whole organism. We identified that Arabidopsis thaliana isoform CPK5 of the plant calcium-dependent protein kinase family becomes rapidly biochemically activated in response to pathogen-associated molecular pattern (PAMP) stimulation. CPK5 signaling resulted in enhanced salicylic acid-mediated resistance to the bacterial pathogen Pst DC3000, differential plant defense gene expression, and synthesis of reactive oxygen species (ROS). Using selected reaction monitoring MS, we identified the plant NADPH oxidase, respiratory burst oxidase homolog D (RBOHD), as an in vivo phosphorylation target of CPK5. Remarkably, CPK5-dependent in vivo phosphorylation of RBOHD occurs on both PAMP- and ROS stimulation. Furthermore, rapid CPK5-dependent biochemical and transcriptional activation of defense reactions at distal sites is compromised in cpk5 and rbohd mutants. Our data not only identify CPK5 as a key regulator of innate immune responses in plants but also support a model of ROS-mediated cell-to-cell communication, where a self-propagating mutual activation circuit consisting of the protein kinase, CPK5, and the NADPH oxidase RBOHD facilitates rapid signal propagation as a prerequisite for defense response activation at distal sites within the plant.
Proceedings of the National Academy of Sciences 05/2013; 110(21). DOI:10.1073/pnas.1221294110 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Calcium (Ca(2+)) is a major second messenger in plant signal transduction mediating stress- and developmental processes. Plant Ca(2+)-dependent protein kinases (CDPKs) are mono-molecular Ca(2+)-sensor/protein kinase effector proteins, which perceive Ca(2+) signals and translate them into protein phosphorylation and thus represent an ideal tool for signal transduction. This review focuses on recent developments in CDPK structural analysis and CDPK in vivo phosphorylation substrate identification. We discuss mechanisms implicated in the in vivo regulation of CDPK activity including Ca(2+) binding to the CDPK EF-hands, Ca(2+)-triggered intra-molecular conformation changes, and CDPK (auto)-phosphorylation. Moreover, we address regulation and integration into signaling cascades of selected members of the plant CDPK family, for which in vivo function and phosphorylation in abiotic and biotic stress signaling has been demonstrated. This article is part of a Special Issue entitled:12th European Symposium on Calcium.
[Show abstract][Hide abstract] ABSTRACT: S-type anion channels are direct targets of abscisic acid (ABA) signaling and contribute to chloride and nitrate release from guard cells, which in turn initiates stomatal closure. SLAC1 was the first component of the guard cell S-type anion channel identified. However, we found that guard cells of Arabidopsis SLAC1 mutants exhibited nitrate conductance. SLAH3 (SLAC1 homolog 3) was also present in guard cells, and coexpression of SLAH3 with the calcium ion (Ca2+)-dependent kinase CPK21 in Xenopus oocytes mediated nitrate-induced anion currents. Nitrate, calcium, and phosphorylation regulated SLAH3 activity. CPK21-dependent SLAH3 phosphorylation and activation were blocked by ABI1, a PP2C-type protein phosphatase that is inhibited by ABA and inhibits the ABA signaling pathway in guard cells. We reconstituted the ABA-stimulated phosphorylation of the SLAH3 amino-terminal domain by CPK21 in vitro by including the ABA receptor-phosphatase complex RCAR1-ABI1 in the reactions. We propose that ABA perception by the complex consisting of ABA receptors of the RCAR/PYR/PYL family and ABI1 releases CPK21 from inhibition by ABI1, and then CPK21 is further activated by an increase in the cytosolic Ca2+ concentration, leading to its phosphorylation of SLAH3. Thus, the identification of SLAH3 as the nitrate-, calcium-, and ABA-sensitive guard cell anion channel provides insights into the relationship among stomatal response to drought, signaling by nitrate, and nitrate metabolism.
[Show abstract][Hide abstract] ABSTRACT: Identification of protein kinase targets and specific inhibition of individual kinase isoforms on the protein level in planta are important techniques to elucidate signal transduction pathways. The use of ATP-binding pocket mutants, the so-called gatekeeper mutants, that accommodate N(6)-enlarged nucleotides and kinase inhibitors has allowed a dramatic increase in kinase isoform selectivity. In this chapter, we describe protocols for the identification and mutation of the gatekeeper residue, radiolabeling of N(6)-modified nucleotides, analysis of protein targets by using [(32)P]-labeled N(6)-modified nucleotides, and in vivo inhibition of kinase activity combined with subsequent molecular readouts. The chapter includes alternative approaches for the described techniques, considerations for other kinases and recommendations toward a setup of a substrate labeling experiment using total cell lysate.
[Show abstract][Hide abstract] ABSTRACT: Calcium-dependent protein kinases (CDPKs) comprise a family of plant serine/threonine protein kinases in which the calcium sensing domain and the kinase effector domain are combined within one molecule. So far, a biological function in abiotic stress signaling has only been reported for few CDPK isoforms, whereas the underlying biochemical mechanism for these CDPKs is still mainly unknown. Here, we show that CPK21 from Arabidopsis thaliana is biochemically activated in vivo in response to hyperosmotic stress. Loss-of-function seedlings of cpk21 are more tolerant to hyperosmotic stress and mutant plants show increased stress responses with respect to marker gene expression and metabolite accumulation. In transgenic Arabidopsis complementation lines in the cpk21 mutant background, in which either CPK21 wild-type, or a full-length enzyme variant carrying an amino-acid substitution were stably expressed, stress responsitivity was restored by CPK21 but not with the kinase inactive variant. The biochemical characterization of in planta synthesized and purified CPK21 protein revealed that within the calcium-binding domain, N-terminal EF1- and EF2-motifs compared to C-terminal EF3- and EF4-motifs differ in their contribution to calcium-regulated kinase activity, suggesting a crucial role for the N-terminal EF-hand pair. Our data provide evidence for CPK21 contributing in abiotic stress signaling and suggest that the N-terminal EF-hand pair is a calcium-sensing determinant controlling specificity of CPK21 function.
[Show abstract][Hide abstract] ABSTRACT: Rice (Oryza sativa) production relies strongly on nitrogen (N) fertilization with urea, but the proteins involved in rice urea metabolism have not yet been characterized. Coding sequences for rice arginase, urease, and the urease accessory proteins D (UreD), F (UreF), and G (UreG) involved in urease activation were identified and cloned. The functionality of urease and the urease accessory proteins was demonstrated by complementing corresponding Arabidopsis (Arabidopsis thaliana) mutants and by multiple transient coexpression of the rice proteins in Nicotiana benthamiana. Secondary structure models of rice (plant) UreD and UreF proteins revealed a possible functional conservation to bacterial orthologs, especially for UreF. Using amino-terminally StrepII-tagged urease accessory proteins, an interaction between rice UreD and urease could be shown. Prokaryotic and eukaryotic urease activation complexes seem conserved despite limited protein sequence conservation for UreF and UreD. In plant metabolism, urea is generated by the arginase reaction. Rice arginase was transiently expressed as a carboxyl-terminally StrepII-tagged fusion protein in N. benthamiana, purified, and biochemically characterized (K(m) = 67 mm, k(cat) = 490 s(-1)). The activity depended on the presence of manganese (K(d) = 1.3 microm). In physiological experiments, urease and arginase activities were not influenced by the external N source, but sole urea nutrition imbalanced the plant amino acid profile, leading to the accumulation of asparagine and glutamine in the roots. Our data indicate that reduced plant performance with urea as N source is not a direct result of insufficient urea metabolism but may in part be caused by an imbalance of N distribution.