Different phosphorylation mechanisms are involved in the activation of sucrose non-fermenting 1 related protein kinases 2 by osmotic stresses and abscisic acid

In Arabidopsis cell suspension, hyperosmotic stresses (mannitol and NaCl) were previously shown to activate nine sucrose non-fermenting 1 related protein kinases 2 (SnRK2s) whereas only five of them were also activated by abscisic acid (ABA) treatment. Here, the possible activation by phosphorylation/dephosphorylation of each kinase was investigated by studying their phosphorylation state after osmotic stress, using the Pro-Q Diamond, a specific dye for phosphoproteins. All the activated kinases were phosphorylated after osmotic stress but the induced phosphorylation changes were clearly different depending on the kinase. In addition, the increase of the global phosphorylation level induced by ABA application was lower, suggesting that different mechanisms may be involved in SnRK2 activation by hyperosmolarity and ABA. On the other hand, SnRK2 kinases remain activated by hyperosmotic stress in ABA-deficient and ABA-insensitive mutants, indicating that SnRK2 osmotic activation is independent of ABA. Moreover, using a mutant form of SnRK2s, a specific serine in the activation loop was shown to be phosphorylated after stress treatments and essential for activity and/or activation. Finally, SnRK2 activity was sensitive to staurosporine, whereas SnRK2 activation by hyperosmolarity or ABA was not, indicating that SnRK2 activation by phosphorylation is mediated by an upstream staurosporine-insensitive kinase, in both signalling pathways. All together, these results indicate that different phosphorylation mechanisms and at least three signalling pathways are involved in the activation of SnRK2 proteins in response to osmotic stress and ABA.     

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

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.     

Arabidopsis Raf‐like kinases act as positive regulators of subclass III SnRK2 in osmostress signaling

Given their sessile nature, land plants must use various mechanisms to manage dehydration under water‐deficit conditions. Osmostress‐induced activation of the SNF1‐related protein kinase 2 (SnRK2) family elicits physiological responses such as stomatal closure to protect plants during drought conditions. With the plant hormone ABA receptors [PYR (pyrabactin resistance)/PYL (pyrabactin resistance‐like)/RCAR (regulatory component of ABA receptors) proteins] and group A protein phosphatases, subclass III SnRK2 also constitutes a core signaling module for ABA, and osmostress triggers ABA accumulation. How SnRK2 is activated through ABA has been clarified, although its activation through osmostress remains unclear. Here, we show that Arabidopsis ABA and abiotic stress‐responsive Raf‐like kinases (AtARKs) of the B3 clade of the mitogen‐activated kinase kinase kinase (MAPKKK) family are crucial in SnRK2‐mediated osmostress responses. Disruption of AtARKs in Arabidopsis results in increased water loss from detached leaves because of impaired stomatal closure in response to osmostress. Our findings obtained in vitro and in planta have shown that AtARKs interact physically with SRK2E, a core factor for stomatal closure in response to drought. Furthermore, we show that AtARK phosphorylates S171 and S175 in the activation loop of SRK2E in vitro and that Atark mutants have defects in osmostress‐induced subclass III SnRK2 activity. Our findings identify a specific type of B3‐MAPKKKs as upstream kinases of subclass III SnRK2 in Arabidopsis. Taken together with earlier reports that ARK is an upstream kinase of SnRK2 in moss, an existing member of a basal land plant lineage, we propose that ARK/SnRK2 module is evolutionarily conserved across 400 million years of land plant evolution for conferring protection against drought.     

Differential activation of the rice sucrose nonfermenting1-related protein kinase2 family by hyperosmotic stress and abscisic acid

To date, a large number of sequences of protein kinases that belong to the sucrose nonfermenting1-related protein kinase2 (SnRK2) family are found in databases. However, only limited numbers of the family members have been characterized and implicated in abscisic acid (ABA) and hyperosmotic stress signaling. We identified 10 SnRK2 protein kinases encoded by the rice (Oryza sativa) genome. Each of the 10 members was expressed in cultured cell protoplasts, and its regulation was analyzed. Here, we demonstrate that all family members are activated by hyperosmotic stress and that three of them are also activated by ABA. Surprisingly, there were no members that were activated only by ABA. The activation was found to be regulated via phosphorylation. In addition to the functional distinction with respect to ABA regulation, dependence of activation on the hyperosmotic strength was different among the members. We show that the relatively diverged C-terminal domain is mainly responsible for this functional distinction, although the kinase domain also contributes to these differences. The results indicated that the SnRK2 protein kinase family has evolved specifically for hyperosmotic stress signaling and that individual members have acquired distinct regulatory properties, including ABA responsiveness by modifying the C-terminal domain.     

NUCLEAR PORE ANCHOR and EARLY IN SHORT DAYS 4 negatively regulate abscisic acid signaling by inhibiting Snf1-related protein kinase2 activity and stability in Arabidopsis

Abscisic acid (ABA) is a key regulator of plant responses to abiotic stresses, such as drought. Abscisic acid receptors and coreceptors perceive ABA to activate Snf1-related protein kinase2s (SnRK2s) that phosphorylate downstream effectors, thereby activating ABA signaling and the stress response. As stress responses come with fitness penalties for plants, it is crucial to tightly control SnRK2 kinase activity to restrict ABA signaling. However, how SnRK2 kinases are inactivated remains elusive. Here, we show that NUCLEAR PORE ANCHOR (NUA), a nuclear pore complex (NPC) component, negatively regulates ABA-mediated inhibition of seed germination and post-germination growth, and drought tolerance in Arabidopsis thaliana. The role of NUA in response to ABA depends on SnRK2.2 and SnRK2.3 for seed germination and on SnRK2.6 for drought. NUA does not directly inhibit the phosphorylation of these SnRK2s or affects their abundance. However, the NUA-interacting protein EARLY IN SHORT DAYS 4 (ESD4), a SUMO protease, negatively regulates ABA signaling by directly interacting with and inhibiting SnRK2 phosphorylation and protein levels. More importantly, we demonstrated that SnRK2.6 can be SUMOylated in vitro, and ESD4 inhibits its SUMOylation. Taken together, we identified NUA and ESD4 as SnRK2 kinase inhibitors that block SnRK2 activity, and reveal a mechanism whereby NUA and ESD4 negatively regulate plant responses to ABA and drought stress possibly through SUMOylation-dependent regulation of SnRK2s.     

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

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.     

The Snf1‐related protein kinases SnRK2.4 and SnRK2.10 are involved in maintenance of root system architecture during salt stress

The sucrose non‐fermenting‐1‐related protein kinase 2 (SnRK2) family represents a unique family of plant‐specific protein kinases implicated in cellular signalling in response to osmotic stress. In our studies, we observed that two class 1 SnRK2 kinases, SnRK2.4 and SnRK2.10, are rapidly and transiently activated in Arabidopsis roots after exposure to salt. Under saline conditions, snrk2.4 knockout mutants had a reduced primary root length, while snrk2.10 mutants exhibited a reduction in the number of lateral roots. The reduced lateral root density was found to be a combinatory effect of a decrease in the number of lateral root primordia and an increase in the number of arrested lateral root primordia. The phenotypes were in agreement with the observed expression patterns of genomic yellow fluorescent protein (YFP) fusions of SnRK2.10 and ‐2.4, under control of their native promoter sequences. SnRK2.10 was found to be expressed in the vascular tissue at the base of a developing lateral root, whereas SnRK2.4 was expressed throughout the root, with higher expression in the vascular system. Salt stress triggered a rapid re‐localization of SnRK2.4–YFP from the cytosol to punctate structures in root epidermal cells. Differential centrifugation experiments of isolated Arabidopsis root proteins confirmed recruitment of endogenous SnRK2.4/2.10 to membranes upon exposure to salt, supporting their observed binding affinity for the phospholipid phosphatidic acid. Together, our results reveal a role for SnRK2.4 and ‐2.10 in root growth and architecture in saline conditions.     

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

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.     

Activation of SnRK2 by Raf-like kinase ARK represents a primary mechanism of ABA and abiotic stress responses

The Raf-like protein kinase abscisic acid (ABA) and abiotic stress-responsive Raf-like kinase (ARK) previously identified in the moss Physcomitrium (Physcomitrella) patens acts as an upstream regulator of subgroup III SNF1-related protein kinase2 (SnRK2), the key regulator of ABA and abiotic stress responses. However, the mechanisms underlying activation of ARK by ABA and abiotic stress for the regulation of SnRK2, including the role of ABA receptor-associated group A PP2C (PP2C-A), are not understood. We identified Ser1029 as the phosphorylation site in the activation loop of ARK, which provided a possible mechanism for regulation of its activity. Analysis of transgenic P. patens ark lines expressing ARK-GFP with Ser1029-to-Ala mutation indicated that this replacement causes reductions in ABA-induced gene expression, stress tolerance, and SnRK2 activity. Immunoblot analysis using an anti-phosphopeptide antibody indicated that ABA treatments rapidly stimulate Ser1029 phosphorylation in the wild type (WT). The phosphorylation profile of Ser1029 in ABA-hypersensitive ppabi1 lacking protein phosphatase 2C-A (PP2C-A) was similar to that in the WT, whereas little Ser1029 phosphorylation was observed in ABA-insensitive ark missense mutant lines. Furthermore, newly isolated ppabi1 ark lines showed ABA-insensitive phenotypes similar to those of ark lines. Therefore, ARK is a primary activator of SnRK2, preceding negative regulation by PP2C-A in bryophytes, which provides a prototype mechanism for ABA and abiotic stress responses in plants.

Phosphorylation in the activation loop of the Raf-like kinase ARK is critical for SNF1-related protein kinase2 regulation during abscisic acid responses in the moss Physcomitrium (Physcomitrella) patens.     

The structure of Arabidopsis thaliana OST1 provides insights into the kinase regulation mechanism in response to osmotic stress

SnRK [SNF1 (sucrose non-fermenting-1)-related protein kinase] 2.6 [open stomata 1 (OST1)] is well characterized at molecular and physiological levels to control stomata closure in response to water-deficit stress. OST1 is a member of a family of 10 protein kinases from Arabidopsis thaliana (SnRK2) that integrates abscisic acid (ABA)-dependent and ABA-independent signals to coordinate the cell response to osmotic stress. A subgroup of protein phosphatases type 2C binds OST1 and keeps the kinase dephosphorylated and inactive. Activation of OST1 relies on the ABA-dependent inhibition of the protein phosphatases type 2C and the subsequent self-phosphorylation of the kinase. The OST1 ABA-independent activation depends on a short sequence motif that is conserved among all the members of the SnRK2 family. However, little is known about the molecular mechanism underlying this regulation. The crystallographic structure of OST1 shows that ABA-independent regulation motif stabilizes the conformation of the kinase catalytically essential α C helix, and it provides the basis of the ABA-independent regulation mechanism for the SnRK2 family of protein kinases.     

ABA-activated SnRK2 protein kinase is required for dehydration stress signaling in Arabidopsis

Protein phosphorylation has pivotal roles in ABA and osmotic stress signaling in higher plants. Two protein phosphatase genes, ABI1 and ABI2, are known to regulate these signaling pathways in Arabidopsis: The identity of ABA-activated protein kinases required for the ABA signaling, however, remains to be elucidated. Here we demonstrate that two protein kinases, p44 and p42, were activated by ABA in Arabidopsis T87 cultured cells, and at least one protein kinase, p44, was activated not only by ABA but also by low humidity in Arabidopsis plants. Analysis of T-DNA knockout mutants and biochemical analysis using a specific antibody revealed that the p44 is encoded by a SnRK2-type protein kinase gene, SRK2E. The srk2e mutation resulted in a wilty phenotype mainly due to loss of stomatal closure in response to a rapid humidity decrease. ABA-inducible gene expression of rd22 and rd29B was suppressed in srk2e. These results show that SRK2E plays an important role in ABA signaling in response to water stress.