Publications by authors named "Grazyna Dobrowolska"

27 Publications

  • Page 1 of 1

The SnRK2.10 kinase mitigates the adverse effects of salinity by protecting photosynthetic machinery.

Plant Physiol 2021 Dec;187(4):2785-2802

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland.

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.
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http://dx.doi.org/10.1093/plphys/kiab438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8644180PMC
December 2021

The Multifaceted Regulation of SnRK2 Kinases.

Cells 2021 08 24;10(9). Epub 2021 Aug 24.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland.

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.
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http://dx.doi.org/10.3390/cells10092180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8465348PMC
August 2021

Regulation of ABA-Non-Activated SNF1-Related Protein Kinase 2 Signaling Pathways by Phosphatidic Acid.

Int J Mol Sci 2020 Jul 15;21(14). Epub 2020 Jul 15.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland.

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.
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http://dx.doi.org/10.3390/ijms21144984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7404309PMC
July 2020

ABA perception is modulated by membrane receptor-like kinases.

J Exp Bot 2020 02;71(4):1210-1214

Institute of Biochemistry and Biophysics, Polish Academy of Science, Warsaw, Poland.

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http://dx.doi.org/10.1093/jxb/erz531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031077PMC
February 2020

Two SnRK2-Interacting Calcium Sensor Isoforms Negatively Regulate SnRK2 Activity by Different Mechanisms.

Plant Physiol 2020 02 7;182(2):1142-1160. Epub 2019 Nov 7.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland.

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 () 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 Ca 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 ::--- or ::--- in the - 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.
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http://dx.doi.org/10.1104/pp.19.00900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997710PMC
February 2020

SnRK2 Protein Kinases and mRNA Decapping Machinery Control Root Development and Response to Salt.

Plant Physiol 2020 01 30;182(1):361-377. Epub 2019 Sep 30.

Plant Cell Biology, University of Amsterdam, Swammerdam Institute for Life Sciences Amsterdam, 1098 XH Amsterdam, The Netherlands

SNF1-RELATED PROTEIN KINASES 2 (SnRK2) are important components of early osmotic and salt stress signaling pathways in plants. The Arabidopsis () 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.
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http://dx.doi.org/10.1104/pp.19.00818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6945840PMC
January 2020

SNF1-Related Protein Kinases SnRK2.4 and SnRK2.10 Modulate ROS Homeostasis in Plant Response to Salt Stress.

Int J Mol Sci 2019 Jan 2;20(1). Epub 2019 Jan 2.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland.

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 , 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.
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http://dx.doi.org/10.3390/ijms20010143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6337402PMC
January 2019

Phosphoproteomic analysis reveals that dehydrins ERD10 and ERD14 are phosphorylated by SNF1-related protein kinase 2.10 in response to osmotic stress.

Plant Cell Environ 2019 03 10;42(3):931-946. Epub 2018 Dec 10.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.

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.
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http://dx.doi.org/10.1111/pce.13465DOI Listing
March 2019

Antisense transcription represses seed dormancy QTL to regulate drought tolerance.

EMBO Rep 2017 12 13;18(12):2186-2196. Epub 2017 Oct 13.

Department of Protein Biosynthesis, Institute of Biochemistry and Biophysics, Warsaw, Poland

Plants have developed multiple strategies to sense the external environment and to adapt growth accordingly. () is a major quantitative trait locus (QTL) for seed dormancy strength in that is reported to be expressed exclusively in seeds. is extensively regulated, with an antisense transcript () suppressing its expression in seeds. Here, we show that shows high levels in mature plants where it suppresses 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 results in constitutive high-level expression, conferring increased drought tolerance, while inactivation of causes enhanced drought sensitivity. The unexpected role of in environmental adaptation of mature plants is separate from its function in seed dormancy regulation. The requirement of to respond to ABA and drought demonstrates that antisense transcription is important for sensing and responding to environmental changes in plants.
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http://dx.doi.org/10.15252/embr.201744862DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5709759PMC
December 2017

Protein phosphatase type 2C PP2CA together with ABI1 inhibits SnRK2.4 activity and regulates plant responses to salinity.

Plant Signal Behav 2016 12;11(12):e1253647

a Institute of Biochemistry and Biophysics, Polish Academy of Sciences , Warsaw , Poland.

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.
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http://dx.doi.org/10.1080/15592324.2016.1253647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5225939PMC
December 2016

Phosphatase ABI1 and okadaic acid-sensitive phosphoprotein phosphatases inhibit salt stress-activated SnRK2.4 kinase.

BMC Plant Biol 2016 06 13;16(1):136. Epub 2016 Jun 13.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106, Warsaw, Poland.

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.
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http://dx.doi.org/10.1186/s12870-016-0817-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4907068PMC
June 2016

Arabidopsis ABA-Activated Kinase MAPKKK18 is Regulated by Protein Phosphatase 2C ABI1 and the Ubiquitin-Proteasome Pathway.

Plant Cell Physiol 2015 Dec 6;56(12):2351-67. Epub 2015 Oct 6.

Department of Biotechnology, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University in Poznań, Umultowska 89, 61-614 Poznań, Poland

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.
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http://dx.doi.org/10.1093/pcp/pcv146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4675898PMC
December 2015

Interplays between nitric oxide and reactive oxygen species in cryptogein signalling.

Plant Cell Environ 2015 Feb 12;38(2):331-48. Epub 2014 Mar 12.

INRA, UMR 1347 Agroécologie, Pôle Mécanisme et Gestion des Interactions Plantes-Microorganismes - ERL CNRS 6300, 17 rue Sully, BP 86510, 21065, Dijon cédex, France.

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.
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http://dx.doi.org/10.1111/pce.12295DOI Listing
February 2015

Nitric oxide and glutathione impact the expression of iron uptake- and iron transport-related genes as well as the content of metals in A. thaliana plants grown under iron deficiency.

Plant Signal Behav 2012 Oct 20;7(10):1246-50. Epub 2012 Aug 20.

Université de Bourgogne, UMR 1347 Agroécologie, Pôle Mécanisme et Gestion des Interactions Plantes-microorganismes, Dijon, France.

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.
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http://dx.doi.org/10.4161/psb.21548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3493405PMC
October 2012

SNF1-related protein kinases type 2 are involved in plant responses to cadmium stress.

Plant Physiol 2012 Oct 10;160(2):868-83. Epub 2012 Aug 10.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland.

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.
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http://dx.doi.org/10.1104/pp.112.194472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3461561PMC
October 2012

Regulation of wound-responsive calcium-dependent protein kinase from maize (ZmCPK11) by phosphatidic acid.

Acta Biochim Pol 2011 12;58(4):589-95. Epub 2011 Dec 12.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland.

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.
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May 2012

SnRK2 protein kinases--key regulators of plant response to abiotic stresses.

OMICS 2011 Dec 2;15(12):859-72. Epub 2011 Dec 2.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.

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.
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http://dx.doi.org/10.1089/omi.2011.0091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3241737PMC
December 2011

SNF1-related protein kinases 2 are negatively regulated by a plant-specific calcium sensor.

J Biol Chem 2011 Feb 22;286(5):3429-41. Epub 2010 Nov 22.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, ul. Pawińskiego 5a, 02-106, Poland.

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.
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http://dx.doi.org/10.1074/jbc.M110.115535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3030349PMC
February 2011

Regulation of Nicotiana tabacum osmotic stress-activated protein kinase and its cellular partner GAPDH by nitric oxide in response to salinity.

Biochem J 2010 Jul;429(1):73-83

UMR INRA 1088/CNRS 5184/Université de Bourgogne, Plante-Microbe-Environnement, 17 rue Sully, 21065 Dijon cedex, France.

Several studies focusing on elucidating the mechanism of NO (nitric oxide) signalling in plant cells have highlighted that its biological effects are partly mediated by protein kinases. The identity of these kinases and details of how NO modulates their activities, however, remain poorly investigated. In the present study, we have attempted to clarify the mechanisms underlying NO action in the regulation of NtOSAK (Nicotiana tabacum osmotic stress-activated protein kinase), a member of the SNF1 (sucrose non-fermenting 1)-related protein kinase 2 family. We found that in tobacco BY-2 (bright-yellow 2) cells exposed to salt stress, NtOSAK is rapidly activated, partly through a NO-dependent process. This activation, as well as the one observed following treatment of BY-2 cells with the NO donor DEA/NO (diethylamine-NONOate), involved the phosphorylation of two residues located in the kinase activation loop, one being identified as Ser158. Our results indicate that NtOSAK does not undergo the direct chemical modifications of its cysteine residues by S-nitrosylation. Using a co-immunoprecipitation-based strategy, we identified several proteins present in immunocomplex with NtOSAK in salt-treated cells including the glycolytic enzyme GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Our results indicate that NtOSAK directly interacts with GAPDH in planta. Furthermore, in response to salt, GAPDH showed a transient increase in its S-nitrosylation level which was correlated with the time course of NtOSAK activation. However, GADPH S-nitrosylation did not influence its interaction with NtOSAK and did not have an impact on the activity of the protein kinase. Taken together, the results support the hypothesis that NtOSAK and GAPDH form a cellular complex and that both proteins are regulated directly or indirectly by NO.
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http://dx.doi.org/10.1042/BJ20100492DOI Listing
July 2010

A novel splicing variant encoding putative catalytic alpha subunit of maize protein kinase CK2.

Physiol Plant 2009 Jul 22;136(3):251-63. Epub 2009 May 22.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland.

A cDNA highly homologous to the known catalytic alpha subunit of protein kinase CK2 was cloned from maize (Zea mays). It was designated ZmCK2alpha-4 (accession no. AAF76187). Sequence analysis shows that ZmCK2alpha-4 and the previously identified ZmCK2alpha-1 (accession no. X61387) are transcribed from the same gene, ZmPKCK2AL (accession no. Y11649), but at different levels in various maize organs and at different stages of development. The cDNA encoding ZmCK2alpha-4 has three potential translation initiation sites. The three putative variants of ZmCK2alpha-4 were expressed in Escherichia coli as GST-fusion proteins and purified from bacterial extracts. In contrast to the previously characterized ZmCK2alphas, the obtained GST:ZmCK2alpha-4 proteins were catalytically inactive as monomers or in the presence of equimolar amounts of the human CK2beta. However, GST:ZmCK2alpha-4 did phosphorylate casein in the presence of a large excess of the beta subunit. The activity of ZmCK2alpha-4 toward casein could also be stimulated by increasing ATP concentration. Modeling studies have shown that there is no interaction between the N-terminal segment of ZmCK2alpha-4 and the activation loop responsible for constitutive catalytic activity of CK2alpha. Preliminary results suggest that ZmCK2alpha-4 may function as a negative regulator of other CK2s, and at certain circumstances as a holoenzyme which catalytic activity is stimulated by specific regulatory subunit(s).
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http://dx.doi.org/10.1111/j.1399-3054.2009.01238.xDOI Listing
July 2009

Nitric oxide in plants: production and cross-talk with Ca2+ signaling.

Mol Plant 2008 Mar 31;1(2):218-28. Epub 2007 Oct 31.

Unité Mixte de Recherche INRA 1088/CNRS 5184/Université de Bourgogne, Plante-Microbe-Environnement, 17 rue Sully, BP 86510, 21065 Dijon cedex, France.

Nitric oxide (NO) is a diatomic gas that performs crucial functions in a wide array of physiological processes in animals. The past several years have revealed much about its roles in plants. It is well established that NO is synthesized from nitrite by nitrate reductase (NR) and via chemical pathways. There is increasing evidence for the occurrence of an alternative pathway in which NO production is catalysed from L-arginine by a so far non-identified enzyme. Contradictory results have been reported regarding the respective involvement of these enzymes in specific physiological conditions. Although much remains to be proved, we assume that these inconsistencies can be accounted for by the limited specificity of the pharmacological agents used to suppress NO synthesis but also by the reduced content of L-arginine as well as the inactivity of nitrate-permeable anion channels in nitrate reductase- and/or nitrate/nitrite-deficient plants. Another unresolved issue concerns the molecular mechanisms underlying NO effects in plants. Here, we provide evidence that the second messenger Ca2+, as well as protein kinases including MAPK and SnRK2, are very plausible mediators of the NO signals. These findings open new perspectives about NO-based signaling in plants.
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http://dx.doi.org/10.1093/mp/ssm016DOI Listing
March 2008

Nitric oxide signalling in plants: interplays with Ca2+ and protein kinases.

J Exp Bot 2008 22;59(2):155-63. Epub 2008 Jan 22.

Unité Mixte de Recherche INRA 1088/CNRS 5184/Université de Bourgogne, Plante-Microbe-Environnement, 17 rue Sully, BP 86510, F-21065 Dijon cedex, France.

Much attention has been paid to nitric oxide (NO) research since its discovery as a physiological mediator of plant defence responses. In recent years, newer roles have been attributed to NO, ranging from root development to stomatal closure. The molecular mechanisms underlying NO action in plants are just begun to emerge. The currently available data illustrate that NO can directly influence the activity of target proteins through nitrosylation and has the capacity to act as a Ca2+-mobilizing intracellular messenger. The interplay between NO and Ca2+ has important functional implications, expanding and enriching the possibilities for modulating transduction processes. Furthermore, protein kinases regulated through NO-dependent mechanisms are being discovered, offering fresh perspective on processes such as stress tolerance.
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http://dx.doi.org/10.1093/jxb/erm197DOI Listing
April 2008

Nicotiana tabacum osmotic stress-activated kinase is regulated by phosphorylation on Ser-154 and Ser-158 in the kinase activation loop.

J Biol Chem 2006 Nov 15;281(45):34299-311. Epub 2006 Sep 15.

Department of Plant Biochemistry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, ul. Pawińskiego 5a, 02-106 Warsaw, Poland.

NtOSAK (Nicotiana tabacum osmotic stress-activated protein kinase), a member of the SnRK2 subfamily, is activated rapidly in response to hyperosmotic stress. Our previous results as well as data presented by others indicate that phosphorylation is involved in activation of SnRK2 kinases. Here, we have mapped the regulatory phosphorylation sites of NtOSAK by mass spectrometry with collision-induced peptide fragmentation. We show that active NtOSAK, isolated from NaCl-treated tobacco BY-2 cells, is phosphorylated on Ser-154 and Ser-158 in the kinase activation loop. Prediction of the NtOSAK three-dimensional structure indicates that phosphorylation of Ser-154 and Ser-158 triggers changes in enzyme conformation resulting in its activation. The involvement of Ser-154 and Ser-158 phosphorylation in regulation of NtOSAK activity was confirmed by site-directed mutagenesis of NtOSAK expressed in bacteria and in maize protoplasts. Our data reveal that phosphorylation of Ser-158 is essential for NtOSAK activation, whereas phosphorylation of Ser-154 most probably facilitates Ser-158 phosphorylation. The time course of NtOSAK phosphorylation on Ser-154 and Ser-158 in BY-2 cells subjected to osmotic stress correlates with NtOSAK activity, indicating that NtOSAK is regulated by reversible phosphorylation of these residues in vivo. Importantly, Ser-154 and Ser-158 are conserved in all SnRK2 subfamily members, suggesting that phosphorylation at these sites may be a general mechanism for SnRK2 activation.
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http://dx.doi.org/10.1074/jbc.M601977200DOI Listing
November 2006

Mechanisms of nitric-oxide-induced increase of free cytosolic Ca2+ concentration in Nicotiana plumbaginifolia cells.

Free Radic Biol Med 2006 Apr 6;40(8):1369-76. Epub 2006 Jan 6.

Unité Mixte de Recherche INRA 1088/CNRS 5184/Université de Bourgogne, Plante-Microbe-Environnement, Dijon, France.

In this study, we investigated a role for nitric oxide (NO) in mediating the elevation of the free cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in plants using Nicotiana plumbaginifolia cells expressing the Ca(2+) reporter apoaequorin. Hyperosmotic stress induced a fast increase of [Ca(2+)](cyt) which was strongly reduced by pretreating cell suspensions with the NO scavenger carboxy PTIO, indicating that NO mediates [Ca(2+)](cyt) changes in plant cells challenged by abiotic stress. Accordingly, treatment of transgenic N. plumbaginifolia cells with the NO donor diethylamine NONOate was followed by a transient increase of [Ca(2+)](cyt) sensitive to plasma membrane Ca(2+) channel inhibitors and antagonist of cyclic ADP ribose. We provided evidence that NO might activate plasma membrane Ca(2+) channels by inducing a rapid and transient plasma membrane depolarization. Furthermore, NO-induced elevation of [Ca(2+)](cyt) was suppressed by the kinase inhibitor staurosporine, suggesting that NO enhances [Ca(2+)](cyt) by promoting phosphorylation-dependent events. This result was further supported by the demonstration that the NO donor induced the activation of a 42-kDa protein kinase which belongs to SnRK2 families and corresponds to Nicotiana tabacum osmotic-stress-activated protein kinase (NtOSAK). Interestingly, NtOSAK was activated in response to hyperosmotic stress through a NO-dependent process, supporting the hypothesis that NO also promotes protein kinase activation during physiological processes.
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http://dx.doi.org/10.1016/j.freeradbiomed.2005.12.006DOI Listing
April 2006

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

Plant Physiol 2005 Dec 18;139(4):1970-83. Epub 2005 Nov 18.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland.

Using protein sequence data obtained from a calcium- and phospholipid-regulated protein kinase purified from maize (Zea mays), we isolated a cDNA encoding a calcium-dependent protein kinase (CDPK), which we designated ZmCPK11. The deduced amino acid sequence of ZmCPK11 includes the sequences of all the peptides obtained from the native protein. The ZmCPK11 sequence contains the kinase, autoregulatory, and calmodulin-like domains typical of CDPKs. Transcripts for ZmCPK11 were present in every tested organ of the plant, relatively high in seeds and seedlings and lower in stems, roots, and leaves. In leaves, kinase activity and ZmCPK11 mRNA accumulation were stimulated by wounding. The level of ZmCPK11 is also increased in noninjured neighboring leaves. The results suggest that the maize protein kinase is involved in a systemic response to wounding. Bacterially expressed glutathione S-transferase (GST)-ZmCPK11 was catalytically active in a calcium-dependent manner. Like the native enzyme, GST-ZmCPK11 was able to phosphorylate histone III-S and Syntide 2. Phosphorylation of histone was stimulated by phosphatidylserine, phosphatidylinositol, and phosphatidic acid, whereas phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, diolein, and cardiolipin did not increase the enzymatic activity. Autophosphorylation of GST-ZmCPK11 was stimulated by calcium and by phosphatidic acid and, to a lesser extent, by phosphatidylserine. Phosphatidylcholine did not affect autophosphorylation. These data unequivocally identify the maize phospholipid- and calcium-regulated protein kinase, which has protein kinase C-like activity, as a CDPK, and emphasize the potential that other CDPKs are regulated by phospholipids in addition to calcium.
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http://dx.doi.org/10.1104/pp.105.066472DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1310574PMC
December 2005

Biochemical characterization of the tobacco 42-kD protein kinase activated by osmotic stress.

Plant Physiol 2004 Oct 1;136(2):3255-65. Epub 2004 Oct 1.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland.

In tobacco (Nicotiana tabacum), hyperosmotic stress induces rapid activation of a 42-kD protein kinase, referred to as Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK). cDNA encoding the kinase was cloned and, based on the predicted amino acid sequence, the enzyme was assigned to the SNF1-related protein kinase type 2 (SnRK2) family. The identity of the enzyme was confirmed by immunoprecipitation of the active kinase from tobacco cells subjected to osmotic stress using antibodies raised against a peptide corresponding to the C-terminal sequence of the kinase predicted from the cloned cDNA. A detailed biochemical characterization of NtOSAK purified from stressed tobacco cells was performed. Our results show that NtOSAK is a calcium-independent Ser/Thr protein kinase. The sequence of putative phosphorylation sites recognized by NtOSAK, predicted by the computer program PREDIKIN, resembled the substrate consensus sequence defined for animal and yeast (Saccharomyces cerevisiae) AMPK/SNF1 kinases. Our experimental data confirmed these results, as various targets for AMPK/SNF1 kinases were also efficiently phosphorylated by NtOSAK. A range of protein kinase inhibitors was tested as potential modulators of NtOSAK, but only staurosporine, a rather nonspecific protein kinase inhibitor, was found to abolish the enzyme activity. In phosphorylation reactions, NtOSAK exhibited a preference for Mg(2+) over Mn(2+) ions and an inability to use GTP instead of ATP as a phosphate donor. The enzyme activity was not modulated by 5'-AMP. To our knowledge, these results represent the first detailed biochemical characterization of a kinase of the SnRK2 family.
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http://dx.doi.org/10.1104/pp.104.046151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC523384PMC
October 2004

Characterization of dual specificity protein kinase from maize seedlings.

Acta Biochim Pol 2004 ;51(3):635-47

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.

A protein kinase of 57 kDa, able to phosphorylate tyrosine in synthetic substrates pol(Glu4,Tyr1) and a fragment of Src tyrosine kinase, was isolated and partly purified from maize seedlings (Zea mays). The protein kinase was able to phosphorylate exogenous proteins: enolase, caseins, histones and myelin basic protein. Amino acid analysis of phosphorylated casein and enolase, as well as of phosphorylated endogenous proteins, showed that both Tyr and Ser residues were phosphorylated. Phosphotyrosine was also immunodetected in the 57 kDa protein fraction. In the protein fraction there are present 57 kDa protein kinase and enolase. This co-purification suggests that enolase can be an endogenous substrate of the kinase. The two proteins could be resolved by two-dimensional electrophoresis. Specific inhibitors of typical protein-tyrosine kinases had essentially no effect on the activity of the maize enzyme. Staurosporine, a nonspecific inhibitor of protein kinases, effectively inhibited the 57 kDa protein kinase. Also, poly L-lysine and heparin inhibited tyrosine phosphorylation by 57 kDa maize protein kinase. The substrate and inhibitor specificities of the 57 kDa maize protein kinase phosphorylating tyrosine indicate that it is a novel plant dual-specificity protein kinase.
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http://dx.doi.org/045103635DOI Listing
March 2005
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