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.

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 StressActivated 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.Cadmium is one of the most toxic soil pollutants. Cadmium ions accumulate in plants and affect, via the food chain, animal and human health. In plants, cadmium is taken up by roots and is transported to aerial organs, leading to chromosomal aberrations, growth reduction, and inhibition of photosynthesis, transpiration, nitrogen metabolism, nutrient and water uptake, eventually causing plant death (for review, see DalCorso et al., 2008). Plants are challenged not only by cadmium ions themselves, but also by Cd 2+ -induced harmful effects including oxidative stress (Schützendübel et al., 2001;Olmos et al., 2003;Cho and Seo, 2005;Sharma and Dietz, 2009). The extent of the detrimental effects on plant growth and metabolism depends on the level of cadmium ions present in the surrounding environment and on the plant's sensitivity to heavy metal stress.Tolerant plants avoid heavy metal uptake and/or induce the expression of genes encoding products involved, directly or indirectly, in heavy metal binding and removal from potentially sensitive sites, by sequestration or efflux (Clemens, 2006). The best-characterized heavy metal binding ligands in plants are thiol-containing compounds metallothioneins and phytochelatins (PCs), whose production is stimulated by Cd 2+. PC...

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.

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.Plants respond to environmental stresses by induction of various defense mechanisms. Stress signals are recognized and transmitted to different cellular compartments by specialized signaling pathways in which protein kinases and phosphatases are key components. The SnRK2 family members are plant-specific kinases considered as important regulators of plant response to abiotic stresses. Ten members of the SnRK2 family have been identified in both Arabidopsis thaliana and Oryza sativa (1, 2). All of them, except SnRK2.9 from A. thaliana, were shown by transient expression in protoplasts to be rapidly activated by treatment with different osmolytes, such as sucrose, mannitol, sorbitol, or NaCl and some of them also by abscisic acid (ABA), 3 suggesting that these kinases are involved in a general response to osmotic stress (1-3). It was also reported that in tobacco BY-2 cells Nicotiana tabacum osmotic stress-activated protein kinase (NtOSAK), a member of the SnRK2 subfamily, is activated rapidly in response to hyperosmotic stress (4 -6).Ample data indicate that SnRK2s are positive regulators of plant response to drought. ABA-activated protein kinase (AAPK) is activated by ABA in guard cells of fava bean (Vicia faba) in response to drought and is involved in the regula...