Grażyna Dobrowolska

Osmotic Stress Induces Rapid Activation of a Salicylic Acid–Induced Protein Kinase and a Homolog of Protein Kinase ASK1 in Tobacco Cells

In tobacco cells, osmotic stress induced the rapid activation of two protein kinases that phosphorylate myelin basic protein. Immunological studies demonstrated that the 48-kD kinase is the salicylic acid–induced protein kinase (SIPK), a member of the mitogen-activated protein kinase family. SIPK was activated 5 to 10 min after the cells were exposed to osmotic stresses, and its activity persisted for ∼30 min. In contrast, the 42-kD kinase was activated within 1 min after osmotic stress, and its activity was maintained for ∼2 hr. Moreover, in addition to myelin basic protein, the 42-kD kinase phosphorylated casein and two transcription factors, c-Jun and ATF-2. This latter enzyme was inactivated by a serine/threonine–specific phosphatase but, unlike SIPK, was not affected by a tyrosine-specific phosphatase. After the 42-kD kinase was purified to apparent homogeneity, tryptic peptide analysis indicated that it is a homolog of Arabidopsis serine/threonine kinase1 (ASK1).

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

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.

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

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.

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

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.

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

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 Mg2+ over Mn2+ 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.

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

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 Cd2+ 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 Cd2+ treatment. Our data show significantly lower Cd2+-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.

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

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.

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

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.

Polypeptides from maize seedlings with protein kinase functions

Abstract Low molecular weight protein kinase from maize seedlings was isolated. The molecular weight of the enzyme was below 10 000. The enzyme preferentially utilized ATP and casein as donor and acceptor of phosphorus, respectively. Casein kinase was a cyclic nucleotide-independent, heparin-sensitive enzyme. The low molecular weight casein kinase was resolved into basic, neutral and slightly acidic peaks of activity by chromatofocussing.

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

ABSTRACT 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.