RESUMO
Remorins are a family of multigenic plasma membrane phosphoproteins involved in biotic and abiotic plant interaction mechanisms, partnering in molecular signaling cascades. Signaling activity of remorins depends on their phosphorylation states and subsequent clustering into nanosized membrane domains. The presence of a coiled-coil domain and a C-terminal domain is crucial to anchor remorins to negatively charged membrane domains; however, the exact role of the N-terminal intrinsically disordered domain (IDD) on protein clustering and lipid interactions is largely unknown. Here, we combine chemical biology and imaging approaches to study the partitioning of group 1 remorin into anionic model membranes mimicking the inner leaflet of the plant plasma membrane. Using reconstituted membranes containing a mix of saturated and unsaturated phosphatidylcholine, phosphatidylinositol phosphates, and sterol, we investigate the clustering of remorins to the membrane and monitor the formation of nanosized membrane domains. REM1.3 promoted membrane nanodomain organization on the exposed external leaflet of both spherical lipid vesicles and flat supported lipid bilayers. Our results reveal that REM1.3 drives a mechanism allowing lipid reorganization, leading to the formation of remorin-enriched nanodomains. Phosphorylation of the N-terminal IDD by the calcium protein kinase CPK3 influences this clustering and can lead to the formation of smaller and more disperse domains. Our work reveals the phosphate-dependent involvement of the N-terminal IDD in the remorin-membrane interaction process by driving structural rearrangements at lipid-water interfaces.
Assuntos
Proteínas de Transporte , Proteínas de Plantas , Proteínas de Transporte/metabolismo , Proteínas de Plantas/química , Membrana Celular/metabolismo , Plantas/metabolismo , Bicamadas Lipídicas/metabolismoRESUMO
MAPKs are universal eukaryotic signaling factors whose functioning is assumed to depend on the recognition of a common docking motif (CD) by its activators, substrates, and inactivators. We studied the role of the CD domain of Arabidopsis MPK4 by performing interaction studies and determining the ligand-bound MPK4 crystal structure. We revealed that the CD domain of MPK4 is essential for interaction and activation by its upstream MAPKKs MKK1, MKK2, and MKK6. Cys181 in the CD site of MPK4 was shown to become sulfenylated in response to reactive oxygen species in vitro. To test the function of C181 in vivo, we generated wild-type (WT) MPK4-C181, nonsulfenylatable MPK4-C181S, and potentially sulfenylation mimicking MPK4-C181D lines in the mpk4 knockout background. We analyzed the phenotypes in growth, development, and stress responses, revealing that MPK4-C181S has WT activity and complements the mpk4 phenotype. By contrast, MPK4-C181D cannot be activated by upstream MAPKK and cannot complement the phenotypes of mpk4. Our findings show that the CD motif is essential and is required for activation by upstream MAPKK for MPK4 function. Furthermore, growth, development, or immunity functions require upstream activation of the MPK4 protein kinase.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Arabidopsis/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Sistema de Sinalização das MAP Quinases , Arabidopsis/metabolismo , Imunidade Vegetal/genéticaRESUMO
Abiotic and biotic factors cause plant wounding and trigger complex short- and long-term responses at the local and systemic levels. These responses are under the control of complex signaling pathways, which are still poorly understood. Here, we show that the rapid activation of clade-A mitogen-activated protein kinases (MAPKs) MPK3 and MPK6 by wounding depends on the upstream MAPK kinases MKK4 and MKK5 but is independent of jasmonic acid (JA) signaling. In addition, this fast module does not control wound-triggered JA accumulation in Arabidopsis (Arabidopsis thaliana), unlike its orthologs in tobacco. We also demonstrate that a second MAPK module, composed of MKK3 and the clade-C MAPKs MPK1/2/7, is activated by wounding in a MKK4/5-independent manner. We provide evidence that the activation of this MKK3-MPK1/2/7 module occurs mainly through wound-induced JA production via the transcriptional regulation of upstream clade-III MAP3Ks, particularly MAP3K14. We show that mkk3 mutant plants are more susceptible to herbivory from larvae of the generalist lepidopteran herbivore Spodoptera littoralis, indicating that the MKK3-MPK1/2/7 module is involved in counteracting insect feeding.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ciclopentanos/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Cinética , Proteínas Quinases Ativadas por Mitógeno/genética , Oxilipinas/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Spodoptera/patogenicidade , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Nutrient signalling integrates and coordinates gene expression, metabolism and growth. However, its primary molecular mechanisms remain incompletely understood in plants and animals. Here we report unique Ca2+ signalling triggered by nitrate with live imaging of an ultrasensitive biosensor in Arabidopsis leaves and roots. A nitrate-sensitized and targeted functional genomic screen identifies subgroup III Ca2+-sensor protein kinases (CPKs) as master regulators that orchestrate primary nitrate responses. A chemical switch with the engineered mutant CPK10(M141G) circumvents embryo lethality and enables conditional analyses of cpk10 cpk30 cpk32 triple mutants to define comprehensive nitrate-associated regulatory and developmental programs. Nitrate-coupled CPK signalling phosphorylates conserved NIN-LIKE PROTEIN (NLP) transcription factors to specify the reprogramming of gene sets for downstream transcription factors, transporters, nitrogen assimilation, carbon/nitrogen metabolism, redox, signalling, hormones and proliferation. Conditional cpk10 cpk30 cpk32 and nlp7 mutants similarly impair nitrate-stimulated system-wide shoot growth and root establishment. The nutrient-coupled Ca2+ signalling network integrates transcriptome and cellular metabolism with shoot-root coordination and developmental plasticity in shaping organ biomass and architecture.
Assuntos
Amidoidrolases/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Cálcio/metabolismo , Nitratos/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais , Amidoidrolases/genética , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Biomassa , Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Carbono/metabolismo , Reprogramação Celular , Alimentos , Regulação da Expressão Gênica de Plantas , Nitrogênio/metabolismo , Oxirredução , Fosforilação , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas , Proteínas Quinases/química , Proteínas Quinases/genética , Transcrição Gênica , TranscriptomaRESUMO
REMORINs (REMs) are a plant-specific protein family, proposed regulators of membrane-associated molecular assemblies and well-established markers of plasma membrane nanodomains. REMs play a diverse set of functions in plant interactions with pathogens and symbionts, responses to abiotic stresses, hormone signaling and cell-to-cell communication. In this review, we highlight the established and more putative roles of REMs throughout the literature. We discuss the physiological functions of REMs, the mechanisms underlying their nanodomain-organization and their putative role as regulators of nanodomain-associated molecular assemblies. Furthermore, we discuss how REM phosphorylation may regulate their functional versatility. Overall, through data-mining and comparative analysis of the literature, we suggest how to further study the molecular mechanisms underpinning the functions of REMs.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Plantas/metabolismoRESUMO
The accurate control of dormancy release and germination is critical for successful plantlet establishment. Investigations in cereals hypothesized a crucial role for specific MAP kinase (MPK) pathways in promoting dormancy release, although the identity of the MPK involved and the downstream events remain unclear. In this work, we characterized mutants for Arabidopsis thaliana MAP kinase 8 (MPK8). Mpk8 seeds presented a deeper dormancy than wild-type (WT) at harvest that was less efficiently alleviated by after-ripening and gibberellic acid treatment. We identified Teosinte Branched1/Cycloidea/Proliferating cell factor 14 (TCP14), a transcription factor regulating germination, as a partner of MPK8. Mpk8 tcp14 double-mutant seeds presented a deeper dormancy at harvest than WT and mpk8, but similar to that of tcp14 seeds. MPK8 interacted with TCP14 in the nucleus in vivo and phosphorylated TCP14 in vitro. Furthermore, MPK8 enhanced TCP14 transcriptional activity when co-expressed in tobacco leaves. Nevertheless, the stimulation of TCP14 transcriptional activity by MPK8 could occur independently of TCP14 phosphorylation. The comparison of WT, mpk8 and tcp14 transcriptomes evidenced that whereas no effect was observed in dry seeds, mpk8 and tcp14 mutants presented dramatic transcriptomic alterations after imbibition with a sustained expression of genes related to seed maturation. Moreover, both mutants exhibited repression of genes involved in cell wall remodeling and cell cycle G1/S transition. As a whole, this study unraveled a role for MPK8 in promoting seed germination, and suggested that its interaction with TCP14 was critical for regulating key processes required for germination completion.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Germinação/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fatores de Transcrição/metabolismo , Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/genética , Parede Celular/genética , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Giberelinas/farmacologia , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Fosforilação , Dormência de Plantas/fisiologia , Plantas Geneticamente Modificadas , Sementes/efeitos dos fármacos , Sementes/fisiologia , Nicotiana/genética , Fatores de Transcrição/genéticaRESUMO
Plants respond to pathogens through dynamic regulation of plasma membrane-bound signaling pathways. To date, how the plant plasma membrane is involved in responses to viruses is mostly unknown. Here, we show that plant cells sense the Potato virus X (PVX) COAT PROTEIN and TRIPLE GENE BLOCK 1 proteins and subsequently trigger the activation of a membrane-bound calcium-dependent kinase. We show that the Arabidopsis thaliana CALCIUM-DEPENDENT PROTEIN KINASE 3-interacts with group 1 REMORINs in vivo, phosphorylates the intrinsically disordered N-terminal domain of the Group 1 REMORIN REM1.3, and restricts PVX cell-to-cell movement. REM1.3's phospho-status defines its plasma membrane nanodomain organization and is crucial for REM1.3-dependent restriction of PVX cell-to-cell movement by regulation of callose deposition at plasmodesmata. This study unveils plasma membrane nanodomain-associated molecular events underlying the plant immune response to viruses.
Assuntos
Proteínas de Transporte/metabolismo , Membrana Celular/imunologia , Fosfoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Potexvirus/patogenicidade , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas do Capsídeo/fisiologia , Membrana Celular/metabolismo , Movimento Celular , Doenças das Plantas/virologia , Folhas de Planta/genética , Folhas de Planta/imunologia , Plantas Geneticamente Modificadas/virologia , Plasmodesmos/metabolismo , Proteínas Quinases/metabolismoRESUMO
Calcium is a ubiquitous second messenger that mediates plant responses to developmental and environmental cues. Calcium-dependent protein kinases (CDPKs) are key actors of plant signaling that convey calcium signals into physiological responses by phosphorylating various substrates including ion channels, transcription factors and metabolic enzymes. This large diversity of targets confers pivotal roles of CDPKs in shoot and root development, pollen tube growth, stomatal movements, hormonal signaling, transcriptional reprogramming and stress tolerance. On the one hand, specificity in CDPK signaling is achieved by differential calcium sensitivities, expression patterns, subcellular localizations and substrates. On the other hand, CDPKs also target some common substrates to ensure key cellular processes indispensable for plant growth and survival in adverse environmental conditions. In addition, the CDPK-related protein kinases (CRKs) might be closer to some CDPKs than previously anticipated and could contribute to calcium signaling despite their inability to bind calcium. This review highlights the regulatory properties of Arabidopsis CDPKs and CRKs that coordinate their multifaceted functions in development, immunity and abiotic stress responses.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas QuinasesRESUMO
Plant surface-localized pathogen recognition receptors (PRRs) perceive conserved microbial features, termed pathogen-associated molecular patterns (PAMPs), resulting in disease resistance. PAMP perception leads to calcium influx, MAPK activation, a burst of reactive oxygen species (ROS) mediated by RbohD, accumulation of the defense hormone salicylic acid (SA), and callose deposition. Lectin receptor-like kinases (LecRKs) belong to a specific PRR family and are important players in plant innate immunity. Here, we report that LecRK-IX.2 is a positive regulator of PRR-triggered immunity. Pathogen infection activated the transcription of Arabidopsis (Arabidopsis thaliana) LecRK-IX.2, and the LecRK-IX.2 knockout lines exhibited enhanced susceptibility to virulent Pseudomonas syringae pv tomato DC3000. In addition, LecRK-IX.2 is capable of inducing RbohD phosphorylation, likely by recruiting calcium-dependent protein kinases to trigger ROS production in Arabidopsis. Overexpression of LecRK-IX.2 resulted in elevated ROS and SA and enhanced systemic acquired resistance to P. syringae pv tomato DC3000. Our data highlight the importance of LecRKs in plant immune signaling and SA accumulation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Lectinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Ácido Salicílico/metabolismo , Transdução de Sinais , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/microbiologia , Morte Celular , Resistência à Doença , Flagelina/metabolismo , Regulação da Expressão Gênica de Plantas , Modelos Biológicos , Mutação/genética , Fenótipo , Fosforilação , Doenças das Plantas/microbiologia , Imunidade Vegetal , Ligação Proteica , Proteínas Quinases/metabolismo , Pseudomonas syringae/fisiologiaRESUMO
Abscisic acid (ABA) is a major phytohormone involved in important stress-related and developmental plant processes. Recent phosphoproteomic analyses revealed a large set of ABA-triggered phosphoproteins as putative mitogen-activated protein kinase (MAPK) targets, although the evidence for MAPKs involved in ABA signalling is still scarce. Here, we identified and reconstituted in vivo a complete ABA-activated MAPK cascade, composed of the MAP3Ks MAP3K17/18, the MAP2K MKK3 and the four C group MAPKs MPK1/2/7/14. In planta, we show that ABA activation of MPK7 is blocked in mkk3-1 and map3k17mapk3k18 plants. Coherently, both mutants exhibit hypersensitivity to ABA and altered expression of a set of ABA-dependent genes. A genetic analysis further reveals that this MAPK cascade is activated by the PYR/PYL/RCAR-SnRK2-PP2C ABA core signalling module through protein synthesis of the MAP3Ks, unveiling an atypical mechanism for MAPK activation in eukaryotes. Our work provides evidence for a role of an ABA-induced MAPK pathway in plant stress signalling.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Sistema de Sinalização das MAP Quinases/fisiologiaRESUMO
Plant stomata function in innate immunity against bacterial invasion and abscisic acid (ABA) has been suggested to regulate this process. Using genetic, biochemical, and pharmacological approaches, we demonstrate that (i) the Arabidopsis thaliana nine-specific-lipoxygenase encoding gene, LOX1, which is expressed in guard cells, is required to trigger stomatal closure in response to both bacteria and the pathogen-associated molecular pattern flagellin peptide flg22; (ii) LOX1 participates in stomatal defense; (iii) polyunsaturated fatty acids, the LOX substrates, trigger stomatal closure; (iv) the LOX products, fatty acid hydroperoxides, or reactive electrophile oxylipins induce stomatal closure; and (v) the flg22-mediated stomatal closure is conveyed by both LOX1 and the mitogen-activated protein kinases MPK3 and MPK6 and involves salicylic acid whereas the ABA-induced process depends on the protein kinases OST1, MPK9, or MPK12. Finally, we show that the oxylipin and the ABA pathways converge at the level of the anion channel SLAC1 to regulate stomatal closure. Collectively, our results demonstrate that early biotic signaling in guard cells is an ABA-independent process revealing a novel function of LOX1-dependent stomatal pathway in plant immunity.
Assuntos
Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Oxilipinas/metabolismo , Estômatos de Plantas/efeitos dos fármacos , Estômatos de Plantas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Imunidade Vegetal/efeitos dos fármacos , Imunidade Vegetal/genética , Estômatos de Plantas/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Innate immunity represents the first line of inducible defence against microbial infection in plants and animals. In both kingdoms, recognition of pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs, respectively), such as flagellin, initiates convergent signalling pathways involving mitogen-activated protein kinase (MAPK) cascades and global transcriptional changes to boost immunity. Although Ca(2+) has long been recognized as an essential and conserved primary mediator in plant defence responses, how Ca(2+) signals are sensed and relayed into early MAMP signalling is unknown. Using a functional genomic screen and genome-wide gene expression profiling, here we show that four calcium-dependent protein kinases (CDPKs) are Ca(2+)-sensor protein kinases critical for transcriptional reprogramming in plant innate immune signalling. Unexpectedly, CDPKs and MAPK cascades act differentially in four MAMP-mediated regulatory programs to control early genes involved in the synthesis of defence peptides and metabolites, cell wall modifications and redox signalling. Transcriptome profile comparison suggests that CDPKs are the convergence point of signalling triggered by most MAMPs. Double, triple and quadruple cpk mutant plants display progressively diminished oxidative burst and gene activation induced by the 22-amino-acid peptide flg22, as well as compromised pathogen defence. In contrast to negative roles of calmodulin and a calmodulin-activated transcription factor in plant defence, the present study reveals Ca(2+) signalling complexity and demonstrates key positive roles of specific CDPKs in initial MAMP signalling.
Assuntos
Arabidopsis/enzimologia , Arabidopsis/imunologia , Imunidade Inata/imunologia , Proteínas Quinases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Flagelina/química , Flagelina/imunologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genoma de Planta/genética , Sistema de Sinalização das MAP Quinases , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Explosão Respiratória , Transcrição GênicaRESUMO
During infection plants recognize microbe-associated molecular patterns (MAMPs), and this leads to stomatal closure. This study analyzes the molecular mechanisms underlying this MAMP response and its interrelation with ABA signaling. Stomata in intact Arabidopsis thaliana plants were stimulated with the bacterial MAMP flg22, or the stress hormone ABA, by using the noninvasive nanoinfusion technique. Intracellular double-barreled microelectrodes were applied to measure the activity of plasma membrane ion channels. Flg22 induced rapid stomatal closure and stimulated the SLAC1 and SLAH3 anion channels in guard cells. Loss of both channels resulted in cells that lacked flg22-induced anion channel activity and stomata that did not close in response to flg22 or ABA. Rapid flg22-dependent stomatal closure was impaired in plants that were flagellin receptor (FLS2)-deficient, as well as in the ost1-2 (Open Stomata 1) mutant, which lacks a key ABA-signaling protein kinase. By contrast, stomata of the ABA protein phosphatase mutant abi1-1 (ABscisic acid Insensitive 1) remained flg22-responsive. These data suggest that the initial steps in flg22 and ABA signaling are different, but that the pathways merge at the level of OST1 and lead to activation of SLAC1 and SLAH3 anion channels.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Bactérias , Flagelina/metabolismo , Canais Iônicos/metabolismo , Proteínas de Membrana/metabolismo , Estômatos de Plantas/fisiologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/fisiologia , Proteínas de Bactérias/farmacologia , Resistência à Doença/fisiologia , Mutação , Fosfoproteínas Fosfatases/metabolismo , Doenças das Plantas/microbiologia , Proteínas Quinases/metabolismo , Transdução de Sinais , Estresse FisiológicoRESUMO
The hydraulic conductivity of plant roots (Lp(r)) is determined in large part by the activity of aquaporins. Mechanisms occurring at the post-translational level, in particular phosphorylation of aquaporins of the plasma membrane intrinsic protein 2 (PIP2) subfamily, are thought to be of critical importance for regulating root water transport. However, knowledge of protein kinases and phosphatases acting on aquaporin function is still scarce. In the present work, we investigated the Lp(r) of knockout Arabidopsis plants for four Ca(2+)-dependent protein kinases. cpk7 plants showed a 30% increase in Lp(r) because of a higher aquaporin activity. A quantitative proteomic analysis of wild-type and cpk7 plants revealed that PIP gene expression and PIP protein quantity were not correlated and that CPK7 has no effect on PIP2 phosphorylation. In contrast, CPK7 exerts a negative control on the cellular abundance of PIP1s, which likely accounts for the higher Lp(r) of cpk7. In addition, this study revealed that the cellular amount of a few additional proteins including membrane transporters is controlled by CPK7. The overall work provides evidence for CPK7-dependent stability of specific membrane proteins.
Assuntos
Aquaporinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Aquaporinas/genética , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Transporte Biológico , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Mutagênese Insercional , Fosforilação , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Transpiração Vegetal/fisiologia , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteômica , Água/fisiologiaRESUMO
CDPKs (calcium-dependent protein kinases), which contain both calmodulin-like calcium binding and serine/threonine protein kinase domains, are only present in plants and some protozoans. Upon activation by a stimulus, they transduce the signal through phosphorylation cascades to induce downstream responses, including transcriptional regulation. To understand the functional specificities of CDPKs, 14 Arabidopsis CPKs (CDPKs in plants) representative of the three main subgroups were characterized at the biochemical level, using HA (haemagglutinin)-tagged CPKs expressed in planta. Most of them were partially or mainly associated with membranes, in agreement with acylation predictions. Importantly, CPKs displayed highly variable calcium-dependences for their kinase activities: seven CPKs from subgroups 1 and 2 were clearly sensitive to calcium with different intensities, whereas six CPKs from subgroup 3 exhibited low or no calcium sensitivity to two generic substrates. Interestingly, this apparent calcium-independence correlated with significant alterations in the predicted EF-hands of these kinases, although they all bound calcium. The noticeable exception, CPK25, was calcium-independent owing to the absence of functional EF-hands. Taken together, the results of the present study suggest that calcium binding differentially affects CDPK isoforms that may be activated by distinct molecular mechanisms.
Assuntos
Arabidopsis/enzimologia , Cálcio/fisiologia , Proteínas Quinases/metabolismo , Proteínas de Arabidopsis/metabolismo , Calmodulina/metabolismo , Motivos EF Hand , Ativação Enzimática , Isoenzimas/metabolismo , Plantas Geneticamente ModificadasRESUMO
[This corrects the article DOI: 10.3389/fpls.2022.862398.].
RESUMO
Calcium-dependent protein kinases (CPK) are key components of a wide array of signaling pathways, translating stress and nutrient signaling into the modulation of cellular processes such as ion transport and transcription. However, not much is known about CPKs in endomembrane trafficking. Here, we screened for CPKs that impact on root growth and gravitropism, by overexpressing constitutively active forms of CPKs under the control of an inducible promoter in Arabidopsis thaliana. We found that inducible overexpression of an constitutive active CPK30 (CA-CPK30) resulted in a loss of root gravitropism and ectopic auxin accumulation in the root tip. Immunolocalization revealed that CA-CPK30 roots have reduced PIN protein levels, PIN1 polarity defects and impaired Brefeldin A (BFA)-sensitive trafficking. Moreover, FM4-64 uptake was reduced, indicative of a defect in endocytosis. The effects on BFA-sensitive trafficking were not specific to PINs, as BFA could not induce aggregation of ARF1- and CHC-labeled endosomes in CA-CPK30. Interestingly, the interference with BFA-body formation, could be reverted by increasing the extracellular pH, indicating a pH-dependence of this CA-CPK30 effect. Altogether, our data reveal an important role for CPK30 in root growth regulation and endomembrane trafficking in Arabidopsis thaliana.
RESUMO
Calcium signaling mediates most developmental processes and stress responses in plants. Among plant calcium sensors, the calcium-dependent protein kinases display a unique structure harboring both calcium sensing and kinase responding activities. AtCPK5 is an essential member of this family in Arabidopsis that regulates immunity and abiotic stress tolerance. To understand the underlying molecular mechanisms, we implemented a biochemical approach to identify in vivo substrates of AtCPK5. We generated transgenic lines expressing a constitutively active form of AtCPK5 under the control of a dexamethasone-inducible promoter. Lines expressing a kinase-dead version were used as a negative control. By comparing the phosphoproteome of the kinase-active and kinase-dead lines upon dexamethasone treatment, we identified 5 phosphopeptides whose abundance increased specifically in the kinase-active lines. Importantly, we showed that all 5 proteins were phosphorylated in vitro by AtCPK5 in a calcium-dependent manner, suggesting that they are direct targets of AtCPK5. We also detected several interaction patterns between the kinase and the candidates in the cytosol, membranes or nucleus, consistent with the ubiquitous localization of AtCPK5. Finally, we further validated the two phosphosites S245 and S280 targeted by AtCPK5 in the E3 ubiquitin ligase ATL31. Altogether, those results open new perspectives to decipher AtCPK5 biological functions.
Assuntos
Arabidopsis/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de PlantasRESUMO
It is now well established that sphingoid Long Chain Bases (LCBs) are crucial mediators of programmed cell death. In plants, the mycotoxin fumonisin B1 (FB1) produced by the necrotrophic fungus Fusarium moniliforme disrupts the sphingolipid biosynthesis pathway by inhibiting the ceramide synthase leading to an increase in the amount of phytosphingosine (PHS) and dihydrosphingosine (DHS), the two major LCBs in Arabidopsis thaliana. To date, the signaling pathway involved in FB1-induced cell death remains largely uncharacterized. It is also well acknowledged that plant proteases such as papain-like cysteine protease are largely involved in plant immunity. Here, we show that the papain-like cysteine protease RD21 (responsive-to-desiccation-21) is activated in response to PHS and FB1 in Arabidopsis cultured cells and leaves, respectively. Using two allelic null mutants of RD21, and two different PCD bioassays, we demonstrate that the protein acts as a negative regulator of FB1-induced cell death in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Morte Celular/fisiologia , Papaína/metabolismo , Esfingolipídeos/metabolismo , Proteínas de Arabidopsis/genética , Morte Celular/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Calcium-dependent protein kinases undergo a rapid biochemical activation in response to an intracellular Ca increase induced by the PRR-dependent perception of a pathogen-related stimulus. Based on SDS gel resolution, the in-gel kinase assay allows the analysis of multiple in vivo protein samples in parallel, combining the advantage of protein separation according to molecular mass with the activity read-out of a protein kinase assay. It thus enables to follow the transient CDPK activation and inactivation in response to in vivo elicitation with a time-wise resolution. In addition, changes of CDPK phosphorylation activity often correlate with slight shifts in the enzyme's apparent molecular mass, indicating posttranslational modifications and a conformational change of the active enzyme compared to its inactive resting form. These band shifts can be detected by a simple immunoblotting to monitor CDPK activation.