ABSTRACT
Members of the calcium-dependent protein kinase (CDPK/CPK) and SNF-related protein kinase (SnRK) superfamilies are commonly found in plants and some protists. Our knowledge of client specificity of the members of this superfamily is fragmentary. As this family is represented by over 30 members in Arabidopsis thaliana, the identification of kinase-specific and overlapping client relationships is crucial to our understanding the nuances of this large family of kinases as directed towards signal transduction pathways. Herein, we used the kinase client (KiC) assay-a relative, quantitative, high-throughput mass spectrometry-based in vitro phosphorylation assay-to identify and characterize potential CPK/SnRK targets of Arabidopsis. Eight CPKs (1, 3, 6, 8, 17, 24, 28, and 32), four SnRKs (subclass 1 and 2), and PPCK1 and PPCK2 were screened against a synthetic peptide library that contains 2095 peptides and 2661 known phosphorylation sites. A total of 625 in vitro phosphorylation sites corresponding to 203 non-redundant proteins were identified. The most promiscuous kinase, CPK17, had 105 candidate target proteins, many of which had already been discovered. Sequence analysis of the identified phosphopeptides revealed four motifs: LxRxxS, RxxSxxR, RxxS, and LxxxxS, that were significantly enriched among CPK/SnRK clients. The results provide insight into both CPK- and SnRK-specific and overlapping signaling network architectures and recapitulate many known in vivo relationships validating this large-scale approach towards discovering kinase targets.
ABSTRACT
SNF1-related protein kinase 2 s (SnRK2s) are major regulators of plant growth, development and responses to environmental stresses. Together with clade A protein phosphatases of type 2C (PP2C) and REGULATORY COMPONENTS OF ABA RECEPTOR (RCAR also known as PYRABACTIN RESISTANCE1 (PYR1) or PYR1-LIKE (PYL)) soluble abscisic acid (ABA) receptors they form the core of ABA-signaling. Clade A PP2Cs play a negative role in ABA signaling, primarily by inhibiting SnRK2 activity, through direct interaction and dephosphorylation of SnRK2s. Here, we describe two methods, which can be used for monitoring inhibition of the SnRK2 activity by PP2C phosphatases. One of them is an in vitro dephosphorylation assay using SnRK2 as the substrate followed by a classical in-gel kinase-activity assay and the other is immunocomplex kinase-activity assay, which can be applied for analysis of the SnRK2 activity in plant material.
Subject(s)
Arabidopsis Proteins , Abscisic Acid/metabolism , Abscisic Acid/pharmacology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Phosphoprotein Phosphatases/genetics , Phosphoprotein Phosphatases/metabolism , Phosphorylation , Signal Transduction/physiologyABSTRACT
SNF1-Related protein kinases Type 2 (SnRK2) are plant-specific enzymes widely distributed across the plant kingdom. They are key players controlling abscisic acid (ABA)-dependent and ABA-independent signaling pathways in the plant response to osmotic stress. Here we established that SnRK2.4 and SnRK2.10, ABA-nonactivated kinases, are activated in Arabidopsis thaliana rosettes during the early response to salt stress and contribute to leaf growth retardation under prolonged salinity but act by maintaining different salt-triggered mechanisms. Under salinity, snrk2.10 insertion mutants were impaired in the reconstruction and rearrangement of damaged core and antenna protein complexes in photosystem II (PSII), which led to stronger non-photochemical quenching, lower maximal quantum yield of PSII, and lower adaptation of the photosynthetic apparatus to high light intensity. The observed effects were likely caused by disturbed accumulation and phosphorylation status of the main PSII core and antenna proteins. Finally, we found a higher accumulation of reactive oxygen species (ROS) in the snrk2.10 mutant leaves under a few-day-long exposure to salinity which also could contribute to the stronger damage of the photosynthetic apparatus and cause other deleterious effects affecting plant growth. We found that the snrk2.4 mutant plants did not display substantial changes in photosynthesis. Overall, our results indicate that SnRK2.10 is activated in leaves shortly after plant exposure to salinity and contributes to salt stress tolerance by maintaining efficient photosynthesis and preventing oxidative damage.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/physiology , Osmotic Pressure , Photosynthesis/physiology , Protein Kinases/genetics , Salt Stress , Arabidopsis/genetics , Arabidopsis Proteins/metabolism , Plant Leaves/growth & development , Protein Kinases/metabolismABSTRACT
SNF1-related kinases 2 (SnRK2s) are central regulators of plant responses to environmental cues simultaneously playing a pivotal role in the plant development and growth in favorable conditions. They are activated in response to osmotic stress and some of them also to abscisic acid (ABA), the latter being key in ABA signaling. The SnRK2s can be viewed as molecular switches between growth and stress response; therefore, their activity is tightly regulated; needed only for a short time to trigger the response, it has to be induced transiently and otherwise kept at a very low level. This implies a strict and multifaceted control of SnRK2s in plant cells. Despite emerging new information concerning the regulation of SnRK2s, especially those involved in ABA signaling, a lot remains to be uncovered, the regulation of SnRK2s in an ABA-independent manner being particularly understudied. Here, we present an overview of available data, discuss some controversial issues, and provide our perspective on SnRK2 regulation.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Abscisic Acid/metabolism , Osmotic Pressure/physiology , Phosphorylation/physiology , Signal Transduction/physiologyABSTRACT
Phosphatidic acid (PA) is involved in the regulation of plant growth and development, as well as responses to various environmental stimuli. Several PA targets in plant cells were identified, including two SNF1-related protein kinases 2 (SnRK2s), SnRK2.10 and SnRK2.4, which are not activated by abscisic acid (ABA). Here, we investigated the effects of PA on various elements of ABA-non-activated SnRK2 signaling. PA 16:0/18:1 was found to modulate the SnRK2 structure and the phosphorylation of some SnRK2 targets. Conversely, phosphorylation by the ABA-non-activated SnRK2s, of one of such targets, dehydrin Early Responsive to Dehydration 14 (ERD14), affects its interaction with PA and subcellular localization. Moreover, PA 16:0/18:1 modulates the activity and/or localization of negative regulators of the ABA-non-activated SnRK2s, not only of the ABA insensitive 1 (ABI1) phosphatase, which was identified earlier, but also of another protein phosphatase 2C, PP2CA. The activity of both phosphatases was inhibited by about 50% in the presence of 50 µM PA. PA 16:0/18:1 also impacts the phosphorylation and subcellular localization of SnRK2-interacting calcium sensor, known to inhibit SnRK2 activity in a calcium-dependent manner. Thus, PA was found to regulate ABA-non-activated SnRK2 signaling at several levels: the activity, phosphorylation status and/or localization of SnRK2 cellular partners.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Phosphatidic Acids/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Abscisic Acid/metabolism , Arabidopsis Proteins/analysis , Phosphorylation , Protein Interaction Maps , Protein Serine-Threonine Kinases/analysisSubject(s)
Abscisic Acid , Arabidopsis Proteins , Arabidopsis Proteins/genetics , Mutation , Perception , Plant StomataABSTRACT
SNF1-related protein kinases 2 (SnRK2s) are key signaling elements regulating abscisic acid-dependent plant development and responses to environmental stresses. Our previous data showed that the SnRK2-interacting Calcium Sensor (SCS) inhibits SnRK2 activity. Use of alternative transcription start sites located within the Arabidopsis (Arabidopsis thaliana) AtSCS gene results in two in-frame transcripts and subsequently two proteins, that differ only by the sequence position of the N terminus. We previously described the longer AtSCS-A, and now describe the shorter AtSCS-B and compare the two isoforms. The two isoforms differ substantially in their expression profiles in plant organs and in response to environmental stresses, in their calcium binding properties, and in their conformational dynamics in the presence and absence of Ca2+ Only AtSCS-A has the features of a calcium sensor. Both forms inhibit SnRK2 activity, but while AtSCS-A requires calcium for inhibition, AtSCS-B does not. Analysis of Arabidopsis plants stably expressing 35S::AtSCS-A-c-myc or 35S::AtSCS-B-c-myc in the scs-1 knockout mutant background revealed that, in planta, both forms are negative regulators of abscisic acid-induced SnRK2 activity and regulate plant resistance against water deficit. Moreover, the data highlight biochemical, biophysical, and functional properties of EF-hand-like motifs in plant proteins.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Calcium/metabolism , Protein Serine-Threonine Kinases/metabolism , Salt Stress/genetics , Stress, Physiological/genetics , Abscisic Acid/metabolism , Abscisic Acid/pharmacology , Algorithms , Arabidopsis/enzymology , Arabidopsis/genetics , Arabidopsis Proteins/antagonists & inhibitors , Arabidopsis Proteins/genetics , Circular Dichroism , Computer Simulation , Dehydration/genetics , Dehydration/metabolism , Gene Expression Regulation, Plant/drug effects , Gene Expression Regulation, Plant/genetics , Gene Knockout Techniques , Hydrogen Deuterium Exchange-Mass Spectrometry , Models, Chemical , Plants, Genetically Modified , Protein Conformation , Protein Domains , Protein Isoforms/metabolism , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Recombinant Proteins , Stress, Physiological/drug effectsABSTRACT
SNF1-RELATED PROTEIN KINASES 2 (SnRK2) are important components of early osmotic and salt stress signaling pathways in plants. The Arabidopsis (Arabidopsis thaliana) SnRK2 family comprises the abscisic acid (ABA)-activated protein kinases SnRK2.2, SnRK2.3, SnRK2.6, SnRK2.7, and SnRK2.8, and the ABA-independent subclass 1 protein kinases SnRK2.1, SnRK2.4, SnRK2.5, SnRK2.9, and SnRK2.10. ABA-independent SnRK2s act at the posttranscriptional level via phosphorylation of VARICOSE (VCS), a member of the mRNA decapping complex, that catalyzes the first step of 5'mRNA decay. Here, we identified VCS and VARICOSE RELATED (VCR) as interactors and phosphorylation targets of SnRK2.5, SnRK2.6, and SnRK2.10. All three protein kinases phosphorylated Ser-645 and Ser-1156 of VCS, whereas SnRK2.6 and SnRK2.10 also phosphorylated VCS Ser-692 and Ser-680 of VCR. We showed that subclass 1 SnRK2s, VCS, and 5' EXORIBONUCLEASE 4 (XRN4) are involved in regulating root growth under control conditions as well as modulating root system architecture in response to salt stress. Our results suggest interesting patterns of redundancy within subclass 1 SnRK2 protein kinases, with SnRK2.1, SnRK2.5, and SnRK2.9 controlling root growth under nonstress conditions and SnRK2.4 and SnRK2.10 acting mostly in response to salinity. We propose that subclass 1 SnRK2s function in root development under salt stress by affecting the transcript levels of aquaporins, as well as CYP79B2, an enzyme involved in auxin biosynthesis.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , RNA, Messenger/metabolism , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Exoribonucleases/genetics , Exoribonucleases/metabolism , Phosphorylation/drug effects , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , RNA, Messenger/genetics , Salts/pharmacology , Signal Transduction/drug effectsABSTRACT
In response to salinity and various other environmental stresses, plants accumulate reactive oxygen species (ROS). The ROS produced at very early stages of the stress response act as signaling molecules activating defense mechanisms, whereas those produced at later stages in an uncontrolled way are detrimental to plant cells by damaging lipids, DNA, and proteins. Multiple systems are involved in ROS generation and also in ROS scavenging. Their level and activity are tightly controlled to ensure ROS homeostasis and protect the plant against the negative effects of the environment. The signaling pathways responsible for maintaining ROS homeostasis in abiotic stress conditions remain largely unknown. Here, we show that in Arabidopsis thaliana, two abscisic acid- (ABA)-non-activated SNF1-releted protein kinases 2 (SnRK2) kinases, SnRK2.4 and SnRK2.10, are involved in the regulation of ROS homeostasis in response to salinity. They regulate the expression of several genes responsible for ROS generation at early stages of the stress response as well as those responsible for their removal. Moreover, the SnRK2.4 regulate catalase levels and its activity and the level of ascorbate in seedlings exposed to salt stress.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Gene Expression Regulation, Plant , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Reactive Oxygen Species/metabolism , Salt Stress , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Catalase/genetics , Catalase/metabolism , Homeostasis , Protein Kinases/genetics , Protein Serine-Threonine Kinases/geneticsABSTRACT
SNF1-related protein kinases 2 (SnRK2s) regulate the plant responses to abiotic stresses, especially water deficits. They are activated in plants subjected to osmotic stress, and some of them are additionally activated in response to enhanced concentrations of abscisic acid (ABA) in plant cells. The SnRK2s that are activated in response to ABA are key elements of ABA signalling that regulate plant acclimation to environmental stresses and ABA-dependent development. Much less is known about the SnRK2s that are not activated by ABA, albeit several studies have shown that these kinases are also involved in response to osmotic stress. Here, we show that one of the Arabidopsis thaliana ABA-non-activated SnRK2s, SnRK2.10, regulates not only the response to salinity but also the plant sensitivity to dehydration. Several potential SnRK2.10 targets phosphorylated in response to stress were identified by a phosphoproteomic approach, including the dehydrins ERD10 and ERD14. Their phosphorylation by SnRK2.10 was confirmed in vitro. Our data suggest that the phosphorylation of ERD14 within the S-segment is involved in the regulation of dehydrin subcellular localization in response to stress.
Subject(s)
Arabidopsis Proteins/metabolism , Osmotic Pressure , Protein Kinases/metabolism , Arabidopsis/metabolism , Arabidopsis/physiology , Arabidopsis Proteins/physiology , Dehydration/metabolism , Mass Spectrometry , Microscopy, Confocal , Phosphorylation , Plants, Genetically Modified , Protein Kinases/physiology , ProteomicsABSTRACT
Plants have developed multiple strategies to sense the external environment and to adapt growth accordingly. Delay of germination 1 (DOG1) is a major quantitative trait locus (QTL) for seed dormancy strength in Arabidopsis thaliana that is reported to be expressed exclusively in seeds. DOG1 is extensively regulated, with an antisense transcript (asDOG1) suppressing its expression in seeds. Here, we show that asDOG1 shows high levels in mature plants where it suppresses DOG1 expression under standard growth conditions. Suppression is released by shutting down antisense transcription, which is induced by the plant hormone abscisic acid (ABA) and drought. Loss of asDOG1 results in constitutive high-level DOG1 expression, conferring increased drought tolerance, while inactivation of DOG1 causes enhanced drought sensitivity. The unexpected role of DOG1 in environmental adaptation of mature plants is separate from its function in seed dormancy regulation. The requirement of asDOG1 to respond to ABA and drought demonstrates that antisense transcription is important for sensing and responding to environmental changes in plants.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Droughts , Plant Dormancy/genetics , RNA, Antisense/genetics , Seeds/genetics , Arabidopsis/anatomy & histology , Arabidopsis/physiology , Arabidopsis Proteins/metabolism , Gene Expression Regulation, Plant , Germination , Mutation , Plant Dormancy/physiology , Plants, Genetically Modified , Seeds/physiology , Transcription FactorsABSTRACT
Protein phosphatases 2C (PP2Cs) are important regulators of plant responses to abiotic stress. It is established that clade A PP2Cs inhibit ABA-activated SNF1-related protein kinases 2 (SnRK2s). Our recently published results show that ABI1, a member of clade A of PP2C is also a negative regulator of SnRK2.4, a kinase not activated in response to ABA. Here, we show that another member of this clade - PP2CA, interacts with and inhibits SnRK2.4. The salt-induced SnRK2.4/SnRK2.10 activity is higher in abi1-2 pp2ca-1 mutant than in wild type or single abi1 or pp2ca mutants, indicating that both phosphatases are inhibitors of SnRK2.4 and are at least partially redundant. Moreover, PP2CA together with ABI1 and SnRK2.4 regulates root growth in response to salinity.
Subject(s)
Abscisic Acid/pharmacology , Arabidopsis Proteins/metabolism , Phosphoprotein Phosphatases/metabolism , Protein Phosphatase 2C/metabolism , Protein Serine-Threonine Kinases/metabolism , Arabidopsis Proteins/genetics , Gene Expression Regulation, Plant/drug effects , Gene Expression Regulation, Plant/genetics , Osmotic Pressure/physiology , Phosphoprotein Phosphatases/genetics , Protein Binding , Protein Phosphatase 2C/genetics , Protein Serine-Threonine Kinases/genetics , Signal Transduction/genetics , Signal Transduction/physiology , Sodium Chloride/pharmacologyABSTRACT
BACKGROUND: SNF1-related protein kinases 2 (SnRK2s) are key regulators of the plant response to osmotic stress. They are transiently activated in response to drought and salinity. Based on a phylogenetic analysis SnRK2s are divided into three groups. The classification correlates with their response to abscisic acid (ABA); group 1 consists SnRK2s non-activated in response to ABA, group 2, kinases non-activated or weakly activated (depending on the plant species) by ABA treatment, and group 3, ABA-activated kinases. The activity of all SnRK2s is regulated by phosphorylation. It is well established that clade A phosphoprotein phosphatases 2C (PP2Cs) are negative regulators of ABA-activated SnRK2s, whereas regulators of SnRK2s from group 1 remain unidentified. RESULTS: Here, we show that ABI1, a PP2C clade A phosphatase, interacts with SnRK2.4, member of group 1 of the SnRK2 family, dephosphorylates Ser158, whose phosphorylation is needed for the kinase activity, and inhibits the kinase, both in vitro and in vivo. Our data indicate that ABI1 and the kinase regulate primary root growth in response to salinity; the phenotype of ABI1 knockout mutant (abi1td) exposed to salt stress is opposite to that of the snrk2.4 mutant. Moreover, we show that the activity of SnRK2s from group 1 is additionally regulated by okadaic acid-sensitive phosphatase(s) from the phosphoprotein phosphatase (PPP) family. CONCLUSIONS: Phosphatase ABI1 and okadaic acid-sensitive phosphatases of the PPP family are negative regulators of salt stress-activated SnRK2.4. The results show that ABI1 inhibits not only the ABA-activated SnRK2s but also at least one ABA-non-activated SnRK2, suggesting that the phosphatase is involved in the cross talk between ABA-dependent and ABA-independent stress signaling pathways in plants.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Phosphoprotein Phosphatases/metabolism , Protein Serine-Threonine Kinases/metabolism , Abscisic Acid/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Okadaic Acid/metabolism , Phosphoprotein Phosphatases/genetics , Phosphorylation , Phylogeny , Protein Binding , Protein Serine-Threonine Kinases/genetics , Sodium Chloride/metabolismABSTRACT
Phosphorylation and dephosphorylation events play an important role in the transmission of the ABA signal. Although SnRK2 [sucrose non-fermenting1-related kinase2] protein kinases and group A protein phosphatase type 2C (PP2C)-type phosphatases constitute the core ABA pathway, mitogen-activated protein kinase (MAPK) pathways are also involved in plant response to ABA. However, little is known about the interplay between MAPKs and PP2Cs or SnRK2 in the regulation of ABA pathways. In this study, an effort was made to elucidate the role of MAP kinase kinase kinase18 (MKKK18) in relation to ABA signaling and response. The MKKK18 knockout lines showed more vigorous root growth, decreased abaxial stomatal index and increased stomatal aperture under normal growth conditions, compared with the control wild-type Columbia line. In addition to transcriptional regulation of the MKKK18 promoter by ABA, we demonstrated using in vitro and in vivo kinase assays that the kinase activity of MKKK18 was regulated by ABA. Analysis of the cellular localization of MKKK18 showed that the active kinase was targeted specifically to the nucleus. Notably, we identified abscisic acid insensitive 1 (ABI1) PP2C as a MKKK18-interacting protein, and demonstrated that ABI1 inhibited its activity. Using a cell-free degradation assay, we also established that MKKK18 was unstable and was degraded by the proteasome pathway. The rate of MKKK18 degradation was delayed in the ABI1 knockout line. Overall, we provide evidence that ABI1 regulates the activity and promotes proteasomal degradation of MKKK18.
Subject(s)
Abscisic Acid/pharmacology , Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , MAP Kinase Kinase Kinases/metabolism , Phosphoprotein Phosphatases/metabolism , Proteasome Endopeptidase Complex/metabolism , Signal Transduction/drug effects , Ubiquitins/metabolism , Arabidopsis/drug effects , Arabidopsis/genetics , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Enzyme Activation/drug effects , Germination/drug effects , Models, Biological , Mutation/genetics , Phenotype , Plant Roots/drug effects , Plant Roots/growth & development , Plant Stomata/drug effects , Plant Stomata/growth & development , Plants, Genetically Modified , Promoter Regions, Genetic/genetics , Protein Binding/drug effects , Protein Phosphatase 2C , Protein Transport/drug effects , Protoplasts/drug effects , Protoplasts/metabolism , Subcellular Fractions/metabolism , NicotianaABSTRACT
Nitric oxide (NO) has many functions in plants. Here, we investigated its interplays with reactive oxygen species (ROS) in the defence responses triggered by the elicitin cryptogein. The production of NO induced by cryptogein in tobacco cells was partly regulated through a ROS-dependent pathway involving the NADPH oxidase NtRBOHD. In turn, NO down-regulated the level of H2O2. Both NO and ROS synthesis appeared to be under the control of type-2 histone deacetylases acting as negative regulators of cell death. Occurrence of an interplay between NO and ROS was further supported by the finding that cryptogein triggered a production of peroxynitrite (ONOO(-)). Next, we showed that ROS, but not NO, negatively regulate the intensity of activity of the cryptogein-induced protein kinase NtOSAK. Furthermore, using a DNA microarray approach, we identified 15 genes early induced by cryptogein via NO. A part of these genes was also modulated by ROS and encoded proteins showing sequence identity to ubiquitin ligases. Their expression appeared to be negatively regulated by ONOO(-), suggesting that ONOO(-) mitigates the effects of NO and ROS. Finally, we provided evidence that NO required NtRBOHD activity for inducing cell death, thus confirming previous assumption that ROS channel NO through cell death pathways.
Subject(s)
Fungal Proteins/metabolism , Nitric Oxide/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction , Fungal Proteins/pharmacology , Gene Expression Regulation, Plant/drug effects , Genes, Plant , Hydrogen Peroxide/metabolism , Models, Biological , Peroxynitrous Acid/metabolism , Plant Proteins/metabolism , Signal Transduction/drug effects , Suspensions , Nicotiana/cytology , Nicotiana/drug effectsABSTRACT
Cadmium ions are notorious environmental pollutants. To adapt to cadmium-induced deleterious effects plants have developed sophisticated defense mechanisms. However, the signaling pathways underlying the plant response to cadmium are still elusive. Our data demonstrate that SnRK2s (for SNF1-related protein kinase2) are transiently activated during cadmium exposure and are involved in the regulation of plant response to this stress. Analysis of tobacco (Nicotiana tabacum) Osmotic Stress-Activated Protein Kinase activity in tobacco Bright Yellow 2 cells indicates that reactive oxygen species (ROS) and nitric oxide, produced mainly via an l-arginine-dependent process, contribute to the kinase activation in response to cadmium. SnRK2.4 is the closest homolog of tobacco Osmotic Stress-Activated Protein Kinase in Arabidopsis (Arabidopsis thaliana). Comparative analysis of seedling growth of snrk2.4 knockout mutants versus wild-type Arabidopsis suggests that SnRK2.4 is involved in the inhibition of root growth triggered by cadmium; the mutants were more tolerant to the stress. Measurements of the level of three major species of phytochelatins (PCs) in roots of plants exposed to Cd(2+) showed a similar (PC2, PC4) or lower (PC3) concentration in snrk2.4 mutants in comparison to wild-type plants. These results indicate that the enhanced tolerance of the mutants does not result from a difference in the PCs level. Additionally, we have analyzed ROS accumulation in roots subjected to Cd(2+) treatment. Our data show significantly lower Cd(2+)-induced ROS accumulation in the mutants' roots. Concluding, the obtained results indicate that SnRK2s play a role in the regulation of plant tolerance to cadmium, most probably by controlling ROS accumulation triggered by cadmium ions.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Cadmium Chloride/pharmacology , Cadmium/metabolism , Protein Serine-Threonine Kinases/metabolism , Stress, Physiological , Adaptation, Physiological , Arabidopsis/drug effects , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Biological Transport , Cytoplasm/genetics , Cytoplasm/metabolism , Enzyme Activation , Gene Knockout Techniques , Iron/metabolism , Microscopy, Confocal , Mutation , Nitric Oxide/metabolism , Phytochelatins/metabolism , Plant Cells/drug effects , Plant Cells/enzymology , Plant Roots/drug effects , Plant Roots/enzymology , Protein Serine-Threonine Kinases/genetics , Reactive Oxygen Species/metabolism , Signal Transduction , Nicotiana/drug effects , Nicotiana/enzymology , Nicotiana/geneticsABSTRACT
Mounting evidence indicate that nitric oxide (NO) acts as a signaling molecule mediating iron deficiency responses through the upregulation of the expression of iron uptake-related genes. Accordingly, NO donors such as nitrosoglutathione (GSNO) were reported to improve the fitness of plants grown under iron deficiency. Here, we showed that glutathione, a by-product of GSNO, triggered the upregulation of the expression of iron uptake- and transport-related gene and an increase of iron concentration in Arabidopsis thaliana seedlings facing iron deficiency. Furthermore, we provided evidence that under iron deficiency, NO released by GSNO did not improve the root iron concentration but impacted the content of copper. Collectively, our data highlight the complexity of interpreting data based on the use of NO donors when investigating the role of NO in iron homeostasis.
Subject(s)
Arabidopsis/growth & development , Arabidopsis/genetics , Gene Expression Regulation, Plant , Glutathione/metabolism , Iron Deficiencies , Iron/metabolism , Nitric Oxide/metabolism , Arabidopsis/drug effects , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Biological Transport/genetics , Copper/metabolism , Gene Expression Regulation, Plant/drug effects , Genes, Plant/genetics , Glutathione/pharmacology , Iron/pharmacology , Manganese/metabolism , Plant Roots/drug effects , Plant Roots/metabolism , S-Nitrosoglutathione/pharmacology , Zinc/metabolismABSTRACT
The SnRK2 family members are plant-specific serine/threonine kinases involved in plant response to abiotic stresses and abscisic acid (ABA)-dependent plant development. SnRK2s have been classed into three groups; group 1 comprises kinases not activated by ABA, group 2 comprises kinases not activated or activated very weakly by ABA, and group 3 comprises kinases strongly activated by ABA. So far, the ABA-dependent kinases belonging to group 3 have been studied most thoroughly. They are considered major regulators of plant response to ABA. The regulation of the plant response to ABA via SnRK2s pathways occurs by direct phosphorylation of various downstream targets, for example, SLAC1, KAT1, AtRbohF, and transcription factors required for the expression of numerous stress response genes. Members of group 2 share some cellular functions with group 3 kinases; however, their contribution to ABA-related responses is not clear. There are strong indications that they are positive regulators of plant responses to water deficit. Most probably they complement the ABA-dependent kinases in plant defense against environmental stress. So far, data concerning the physiological role of ABA-independent SnRK2s are very limited; it is to be expected they will be studied extensively in the nearest future.
Subject(s)
Plants/enzymology , Protein Serine-Threonine Kinases/metabolism , Stress, Physiological , Gene Expression Regulation, Plant , Phylogeny , Plants/genetics , Protein Serine-Threonine Kinases/classification , Protein Serine-Threonine Kinases/genetics , Signal TransductionABSTRACT
In plant cells, phospholipids are not only membrane components but also act as second messengers interacting with various proteins and regulating diverse cellular processes, including stress signal transduction. Here, we report studies on the effects of various phospholipids on the activity and expression of maize wound-responsive calcium-dependent protein kinase (ZmCPK11). Our results revealed that in leaves treated with n-butanol, a potent inhibitor of phosphatidic acid (PA) synthesis catalyzed by phospholipase D, a significant decrease of ZmCPK11 activity was observed, indicating contribution of PA in the kinase activation. Using lipid binding assays, we demonstrate that among various phospholipids only saturated acyl species (16:0 and 18:0) of phosphatidic acid are able to bind to ZmCPK11. Saturated acyl species of PA are also able to stimulate phosphorylation of exogenous substrates by ZmCPK11 and autophosphorylation of the kinase. The level of ZmCPK11 autophosphorylation is correlated with its enzymatic activity. RT-PCR analysis showed that transcript level of ZmCPK11 in maize leaves increased in response to PA treatment. The influence of PA on the activity and transcript level of ZmCPK11 suggests an involvement of this kinase in a PA-mediated wound signal transduction pathway.
Subject(s)
Gene Expression Regulation, Plant , Phosphatidic Acids/pharmacology , Plant Proteins/metabolism , Protein Kinases/metabolism , Zea mays/enzymology , 1-Butanol/pharmacology , Enzyme Activation , Enzyme Assays , Genes, Plant , Mutagenesis, Site-Directed , Phosphatidic Acids/metabolism , Phospholipase D/genetics , Phospholipase D/metabolism , Phosphorylation , Plant Leaves/drug effects , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Proteins/genetics , Protein Binding , Protein Kinases/genetics , Protoplasts/cytology , Protoplasts/metabolism , RNA, Messenger/analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Stress, Physiological , Time Factors , Transfection , Zea mays/drug effects , Zea mays/geneticsABSTRACT
SNF1-related protein kinases 2 (SnRK2s) are plant-specific enzymes involved in environmental stress signaling and abscisic acid-regulated plant development. Here, we report that SnRK2s interact with and are regulated by a plant-specific calcium-binding protein. We screened a Nicotiana plumbaginifolia Matchmaker cDNA library for proteins interacting with Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK), a member of the SnRK2 family. A putative EF-hand calcium-binding protein was identified as a molecular partner of NtOSAK. To determine whether the identified protein interacts only with NtOSAK or with other SnRK2s as well, we studied the interaction of an Arabidopsis thaliana orthologue of the calcium-binding protein with selected Arabidopsis SnRK2s using a two-hybrid system. All kinases studied interacted with the protein. The interactions were confirmed by bimolecular fluorescence complementation assay, indicating that the binding occurs in planta, exclusively in the cytoplasm. Calcium binding properties of the protein were analyzed by fluorescence spectroscopy using Tb(3+) as a spectroscopic probe. The calcium binding constant, determined by the protein fluorescence titration, was 2.5 ± 0.9 × 10(5) M(-1). The CD spectrum indicated that the secondary structure of the protein changes significantly in the presence of calcium, suggesting its possible function as a calcium sensor in plant cells. In vitro studies revealed that the activity of SnRK2 kinases analyzed is inhibited in a calcium-dependent manner by the identified calcium sensor, which we named SCS (SnRK2-interacting calcium sensor). Our results suggest that SCS is involved in response to abscisic acid during seed germination most probably by negative regulation of SnRK2s activity.