An abscisic acid-independent oxylipin pathway controls stomatal closure and immune defense in Arabidopsis.

Plant stomata function in innate immunity against bacterial invasion and abscisic acid (ABA) has been suggested to regulate this process. Using genetic, biochemical, and pharmacological approaches, we demonstrate that (i) the Arabidopsis thaliana nine-specific-lipoxygenase encoding gene, LOX1, which is expressed in guard cells, is required to trigger stomatal closure in response to both bacteria and the pathogen-associated molecular pattern flagellin peptide flg22; (ii) LOX1 participates in stomatal defense; (iii) polyunsaturated fatty acids, the LOX substrates, trigger stomatal closure; (iv) the LOX products, fatty acid hydroperoxides, or reactive electrophile oxylipins induce stomatal closure; and (v) the flg22-mediated stomatal closure is conveyed by both LOX1 and the mitogen-activated protein kinases MPK3 and MPK6 and involves salicylic acid whereas the ABA-induced process depends on the protein kinases OST1, MPK9, or MPK12. Finally, we show that the oxylipin and the ABA pathways converge at the level of the anion channel SLAC1 to regulate stomatal closure. Collectively, our results demonstrate that early biotic signaling in guard cells is an ABA-independent process revealing a novel function of LOX1-dependent stomatal pathway in plant immunity.

Characterization of Arabidopsis calcium-dependent protein kinases: activated or not by calcium?

CDPKs (calcium-dependent protein kinases), which contain both calmodulin-like calcium binding and serine/threonine protein kinase domains, are only present in plants and some protozoans. Upon activation by a stimulus, they transduce the signal through phosphorylation cascades to induce downstream responses, including transcriptional regulation. To understand the functional specificities of CDPKs, 14 Arabidopsis CPKs (CDPKs in plants) representative of the three main subgroups were characterized at the biochemical level, using HA (haemagglutinin)-tagged CPKs expressed in planta. Most of them were partially or mainly associated with membranes, in agreement with acylation predictions. Importantly, CPKs displayed highly variable calcium-dependences for their kinase activities: seven CPKs from subgroups 1 and 2 were clearly sensitive to calcium with different intensities, whereas six CPKs from subgroup 3 exhibited low or no calcium sensitivity to two generic substrates. Interestingly, this apparent calcium-independence correlated with significant alterations in the predicted EF-hands of these kinases, although they all bound calcium. The noticeable exception, CPK25, was calcium-independent owing to the absence of functional EF-hands. Taken together, the results of the present study suggest that calcium binding differentially affects CDPK isoforms that may be activated by distinct molecular mechanisms.

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 cytosolic/nuclear HSC70 and HSP90 molecular chaperones are important for stomatal closure and modulate abscisic acid-dependent physiological responses in Arabidopsis.

Cytosolic/nuclear molecular chaperones of the heat shock protein families HSP90 and HSC70 are conserved and essential proteins in eukaryotes. These proteins have essentially been implicated in the innate immunity and abiotic stress tolerance in higher plants. Here, we demonstrate that both chaperones are recruited in Arabidopsis (Arabidopsis thaliana) for stomatal closure induced by several environmental signals. Plants overexpressing HSC70-1 or with reduced HSP90.2 activity are compromised in the dark-, CO(2)-, flagellin 22 peptide-, and abscisic acid (ABA)-induced stomatal closure. HSC70-1 and HSP90 proteins are needed to establish basal expression levels of several ABA-responsive genes, suggesting that these chaperones might also be involved in ABA signaling events. Plants overexpressing HSC70-1 or with reduced HSP90.2 activity are hypersensitive to ABA in seed germination assays, suggesting that several chaperone complexes with distinct substrates might tune tissue-specific responses to ABA and the other biotic and abiotic stimuli studied. This study demonstrates that the HSC70/HSP90 machinery is important for stomatal closure and serves essential functions in plants to integrate signals from their biotic and abiotic environments.

Phospho-site mapping, genetic and in planta activation studies reveal key aspects of the different phosphorylation mechanisms involved in activation of SnRK2s.

Snf1-related protein kinases 2 (SnRK2s) are major positive regulators of drought stress tolerance. The kinases of this family are activated by hyperosmotic stress, but only some of them are also responsive to abscisic acid (ABA). Moreover, genetic evidence has indicated the ABA-independence of SnRK2 activation in the fast response to osmotic stress. Although phosphorylation was demonstrated to be crucial for the activation or activity of the kinases of both subgroups, different phosphorylation mechanisms were suggested. Here, using one kinase from each subgroup (SnRK2.6 and SnRK2.10), two phosphorylation sites within the activation loop were identified by mass spectrometry after immunoprecipitation from Arabidopsis cells treated by ABA or osmolarity. By site-directed mutagenesis, the phosphorylation of only one of the two sites was shown to be necessary for the catalytic activity of the kinase, whereas both sites are necessary for the full activation of the two SnRK2s by hyperosmolarity or ABA. Phosphoprotein staining together with two-dimensional PAGE followed by immunoblotting indicated distinct phosphorylation mechanisms of the two kinases. While SnRK2.6 seems to be activated through the independent phosphorylation of these two sites, a sequential process occurs in SnRK2.10, where phosphorylation of one serine is required for the phosphorylation of the other. In addition, a subgroup of protein phosphatases 2C which interact and participate in the regulation of SnRK2.6 do not interact with SnRK2.10. Taken together, our data bring evidence for the involvement of distinct phosphorylation mechanisms in the activation of SnRK2.6 and SnRK2.10, which may be conserved between the two subgroups of SnRK2s depending on their ABA-responsiveness.

Protein phosphatases 2C regulate the activation of the Snf1-related kinase OST1 by abscisic acid in Arabidopsis.

The plant hormone abscisic acid (ABA) orchestrates plant adaptive responses to a variety of stresses, including drought. This signaling pathway is regulated by reversible protein phosphorylation, and genetic evidence demonstrated that several related protein phosphatases 2C (PP2Cs) are negative regulators of this pathway in Arabidopsis thaliana. Here, we developed a protein phosphatase profiling strategy to define the substrate preferences of the HAB1 PP2C implicated in ABA signaling and used these data to screen for putative substrates. Interestingly, this analysis designated the activation loop of the ABA activated kinase OST1, related to Snf1 and AMPK kinases, as a putative HAB1 substrate. We experimentally demonstrated that HAB1 dephosphorylates and deactivates OST1 in vitro. Furthermore, HAB1 and the related PP2Cs ABI1 and ABI2 interact with OST1 in vivo, and mutations in the corresponding genes strongly affect OST1 activation by ABA. Our results provide evidence that PP2Cs are directly implicated in the ABA-dependent activation of OST1 and further suggest that the activation mechanism of AMPK/Snf1-related kinases through the inhibition of regulating PP2Cs is conserved from plants to human.

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.

Involvement of MPK4 in osmotic stress response pathways in cell suspensions and plantlets of Arabidopsis thaliana: activation by hypoosmolarity and negative role in hyperosmolarity tolerance.

Three of the protein kinases activated by hypoosmotic stress in Arabidopsis thaliana cell suspensions were previously characterized [FEBS, 2002, 527, 43-50] as mitogen-activated protein (MAP) kinases and two of them corresponded to Arabidopsis mitogen-activated protein kinase 6 (MPK6) (44 kDa) and MPK3 (39 kDa). The third MAP kinase was identified here to MPK4, using a corresponding specific antibody. Like MPK6 and MPK3, MPK4 activity is clearly inhibited by apigenin and MPK4 activation by hypoosmolarity needs upstream phosphorylation events. Activation of the 3 MAP kinases, MPK3, 4 and 6, was confirmed in plantlets submitted to hypoosmotic stress. The action of a biotic signal, flagellin, was also demonstrated to induce the activations of the 3 MAP kinases. Using the mutant displaying MPK4 gene inactivation, the independence of the MPK3 and MPK6 activations towards the presence of MPK4 was demonstrated, both in hypoosmotic and flagellin signalling pathways. Although MPK4 was not activated by hyperosmolarity in cell suspensions nor in seedlings, a possible negative regulation of hyperosmolarity resistance by MPK4 is suggested, based both on phenotype and downstream gene expression studies.

Identification of nine sucrose nonfermenting 1-related protein kinases 2 activated by hyperosmotic and saline stresses in Arabidopsis thaliana.

Several calcium-independent protein kinases were activated by hyperosmotic and saline stresses in Arabidopsis cell suspension. Similar activation profiles were also observed in seedlings exposed to hyperosmotic stress. One of them was identified to AtMPK6 but the others remained to be identified. They were assumed to belong to the SNF1 (sucrose nonfermenting 1)-related protein kinase 2 (SnRK2) family, which constitutes a plant-specific kinase group. The 10 Arabidopsis SnRK2 were expressed both in cells and seedlings, making the whole SnRK2 family a suitable candidate. Using a family-specific antibody raised against the 10 SnRK2, we demonstrated that these non-MAPK protein kinases activated by hyperosmolarity in cell suspension were SnRK2 proteins. Then, the molecular identification of the involved SnRK2 was investigated by transient expression assays. Nine of the 10 SnRK2 were activated by hyperosmolarity induced by mannitol, as well as NaCl, indicating an important role of the SnRK2 family in osmotic signaling. In contrast, none of the SnRK2 were activated by cold treatment, whereas abscisic acid only activated five of the nine SnRK2. The probable involvement of the different Arabidopsis SnRK2 in several abiotic transduction pathways is discussed.

The MAP kinase kinase NtMEK2 is involved in tobacco pollen germination.

The tobacco ntf4 mitogen-activated protein (MAP) kinase gene (and its encoded protein p45(Ntf4)) is expressed at later stages of pollen maturation. We have found that the highly related MAP kinase SIPK is also expressed in pollen and, like p45(Ntf4), is activated upon pollen hydration. The MAP kinase kinase NtMEK2 activates SIPK, and here we show that it can also activate p45(Ntf4). In an attempt to inhibit the function of both MAP kinases simultaneously we constructed a loss-of-function mutant version of NtMEK2, which, in transient transformation assays, led to an inhibition of germination in the transformed pollen grains. These data indicate that NtMEK2, and by inference its substrates p45(Ntf4) and/or SIPK, are involved in pollen germination.

Different protein kinase families are activated by osmotic stresses in Arabidopsis thaliana cell suspensions. Involvement of the MAP kinases AtMPK3 and AtMPK6.

Five Ca(2+)-independent protein kinases were rapidly activated by hypoosmotic stress, moderate or high hyperosmolarity induced by several osmolytes, sucrose, mannitol or NaCl. Three of these kinases, transiently activated by hypoosmolarity, recognised by anti-phosphorylated mitogen-activated protein (MAP) kinase antibodies, sensitive to a MAP kinase inhibitor and inactivated by the action of a tyrosine phosphatase, corresponded to MAP kinases. Using specific antibodies, two of the MAP kinases were identified as AtMPK6 and AtMPK3. The two other protein kinases, durably activated by high hyperosmolarity, did not belong to the MAP kinase family. Activation of AtMPK6 and AtMPK3 by hypoosmolarity depended on upstream protein kinases sensitive to staurosporine and on calcium influx. In contrast, these two transduction steps were not involved in the activation of the two protein kinases activated by high hyperosmolarity.