Molecular characterization of maize bHLH transcription factor (ZmKS), a new ZmOST1 kinase substrate.

In order to identify potential substrates of the maize kinase in the ABA signalling network, ZmOST1 was used as bait against a library of cDNAs from dehydrated young leaves. A ZmOST1-interactive polypeptide ZmKS (gene locus tag: GRMZM2G114873), showing homology with the Arabidopsis thaliana basic helix-loop-helix (bHLH) DNA-binding transcription factor was identified. Using a comparative genomic approach, the ZmKS corresponding protein was identified as conceptual translated bHLH transcription factor ABA-responsive kinase substrate. ZmKS is localized in the nucleus, shows a potential binding specificity preferentially detectable on cis-acting E-box like heptameric motifs CCACTTG and CAAGTTG, and is phosphorylated by maize protein kinase ZmOST1. ZmKS is expressed in embryo, leaf and root, expression being affected by ABA and osmotic stress. Transgenic Arabidopsis plants, with gain of ZmKS function, show a delay in germination and a transcriptional stomatal opening-facilitator activity, switchover upon ZmKS phosphorylation, suggesting that ZmKS is an ABA-repressed trans-acting activator.

Casein Kinase 2 Negatively Regulates Abscisic Acid-Activated SnRK2s in the Core Abscisic Acid-Signaling Module.

SnRK2 kinases, PP2C phosphatases and the PYR/PYL/RCAR receptors constitute the core abscisic acid (ABA) signaling module that is thought to contain all of the intrinsic properties to self-regulate the hormone signal output. Here we identify Casein Kinase (CK)2 as a novel negative regulator of SnRK2. CK2 phosphorylates a cluster of conserved serines at the ABA box of SnRK2, increasing its binding to PP2C and triggering protein degradation. Consequently, CK2 action has implications on SnRK2 protein levels, as well as kinase activity and its response to abiotic stimuli.

The heat shock factor A4A confers salt tolerance and is regulated by oxidative stress and the mitogen-activated protein kinases MPK3 and MPK6.

Heat shock factors (HSFs) are principal regulators of plant responses to several abiotic stresses. Here, we show that estradiol-dependent induction of HSFA4A confers enhanced tolerance to salt and oxidative agents, whereas inactivation of HSFA4A results in hypersensitivity to salt stress in Arabidopsis (Arabidopsis thaliana). Estradiol induction of HSFA4A in transgenic plants decreases, while the knockout hsfa4a mutation elevates hydrogen peroxide accumulation and lipid peroxidation. Overexpression of HSFA4A alters the transcription of a large set of genes regulated by oxidative stress. In yeast (Saccharomyces cerevisiae) two-hybrid and bimolecular fluorescence complementation assays, HSFA4A shows homomeric interaction, which is reduced by alanine replacement of three conserved cysteine residues. HSFA4A interacts with mitogen-activated protein kinases MPK3 and MPK6 in yeast and plant cells. MPK3 and MPK6 phosphorylate HSFA4A in vitro on three distinct sites, serine-309 being the major phosphorylation site. Activation of the MPK3 and MPK6 mitogen-activated protein kinase pathway led to the transcriptional activation of the HEAT SHOCK PROTEIN17.6A gene. In agreement that mutation of serine-309 to alanine strongly diminished phosphorylation of HSFA4A, it also strongly reduced the transcriptional activation of HEAT SHOCK PROTEIN17.6A. These data suggest that HSFA4A is a substrate of the MPK3/MPK6 signaling and that it regulates stress responses in Arabidopsis.

ABI1 and PP2CA phosphatases are negative regulators of Snf1-related protein kinase1 signaling in Arabidopsis.

Plant survival under environmental stress requires the integration of multiple signaling pathways into a coordinated response, but the molecular mechanisms underlying this integration are poorly understood. Stress-derived energy deprivation activates the Snf1-related protein kinases1 (SnRK1s), triggering a vast transcriptional and metabolic reprogramming that restores homeostasis and promotes tolerance to adverse conditions. Here, we show that two clade A type 2C protein phosphatases (PP2Cs), established repressors of the abscisic acid (ABA) hormonal pathway, interact with the SnRK1 catalytic subunit causing its dephosphorylation and inactivation. Accordingly, SnRK1 repression is abrogated in double and quadruple pp2c knockout mutants, provoking, similarly to SnRK1 overexpression, sugar hypersensitivity during early seedling development. Reporter gene assays and SnRK1 target gene expression analyses further demonstrate that PP2C inhibition by ABA results in SnRK1 activation, promoting SnRK1 signaling during stress and once the energy deficit subsides. Consistent with this, SnRK1 and ABA induce largely overlapping transcriptional responses. Hence, the PP2C hub allows the coordinated activation of ABA and energy signaling, strengthening the stress response through the cooperation of two key and complementary pathways.

The maize OST1 kinase homolog phosphorylates and regulates the maize SNAC1-type transcription factor.

The Arabidopsis kinase OPEN STOMATA 1 (OST1) plays a key role in regulating drought stress signalling, particularly stomatal closure. We have identified and investigated the functions of the OST1 ortholog in Z. mays (ZmOST1). Ectopic expression of ZmOST1 in the Arabidopsis ost1 mutant restores the stomatal closure phenotype in response to drought. Furthermore, we have identified the transcription factor, ZmSNAC1, which is directly phosphorylated by ZmOST1 with implications on its localization and protein stability. Interestingly, ZmSNAC1 binds to the ABA-box of ZmOST1, which is conserved in SnRK2s activated by ABA and is part of the contact site for the negative-regulating clade A PP2C phosphatases. Taken together, our results indicate that ZmSNAC1 is a substrate of ZmOST1 and delineate a novel osmotic stress transcriptional pathway in maize.

Role of plant-specific N-terminal domain of maize CK2ß1 subunit in CK2ß functions and holoenzyme regulation.

Protein kinase CK2 is a highly pleiotropic Ser/Thr kinase ubiquituous in eukaryotic organisms. CK2 is organized as a heterotetrameric enzyme composed of two types of subunits: the catalytic (CK2α) and the regulatory (CK2ß). The CK2ß subunits enhance the stability, activity and specificity of the holoenzyme, but they can also perform functions independently of the CK2 tetramer. CK2ß regulatory subunits in plants differ from their animal or yeast counterparts, since they present an additional specific N-terminal extension of about 90 aminoacids that shares no homology with any previously characterized functional domain. Sequence analysis of the N-terminal domain of land plant CK2ß subunit sequences reveals its arrangement through short, conserved motifs, some of them including CK2 autophosphorylation sites. By using maize CK2ß1 and a deleted version (ΔNCK2ß1) lacking the N-terminal domain, we have demonstrated that CK2ß1 is autophosphorylated within the N-terminal domain. Moreover, the holoenzyme composed with CK2α1/ΔNCK2ß1 is able to phosphorylate different substrates more efficiently than CK2α1/CK2ß1 or CK2α alone. Transient overexpression of CK2ß1 and ΔNCK2ß1 fused to GFP in different plant systems show that the presence of N-terminal domain enhances aggregation in nuclear speckles and stabilizes the protein against proteasome degradation. Finally, bimolecular fluorescence complementation (BiFC) assays show the nuclear and cytoplasmic location of the plant CK2 holoenzyme, in contrast to the individual CK2α/ß subunits mainly observed in the nucleus. All together, our results support the hypothesis that the plant-specific N-terminal domain of CK2ß subunits is involved in the down-regulation of the CK2 holoenzyme activity and in the stabilization of CK2ß1 protein. In summary, the whole amount of data shown in this work suggests that this domain was acquired by plants for regulatory purposes.

Regulation of MAPK signaling and cell death by MAPK phosphatase MKP2.

Mitogen-activated protein kinase (MAPK) pathways play crucial roles in developmental and adaptive responses. Depending on the stimulus, MAPK activation regulates a wide variety of plant cell responses, such as proliferation, differentiation and cell death, which normally require precise spatial and temporal control. In this context, protein phosphatases play important roles by regulating the duration and magnitude of MAPK activities. During infection by non-host and incompatible host microorganisms, MAPK activity can promote a local cell death mechanism called hypersensitivity response (HR), which is part of the plant defence response. HR-like responses require sustained MAPK activity and correlate with oxidative burst. We recently showed that MAPK phosphatase MKP2 positively controls biotic and abiotic stress responses in Arabidopsis. MKP2 interacts with MPK6 in HR-like responses triggered by fungal elicitors, suggesting that MKP2 protein is part of the mechanism involved in MAPK regulation during HR. Here we discuss the interplay of MAPK and MKP2 phosphatase signaling during cell death responses elicited by host-pathogen interactions.

MAPK phosphatase MKP2 mediates disease responses in Arabidopsis and functionally interacts with MPK3 and MPK6.

Mitogen-activated protein kinase (MAPK) cascades have important functions in plant stress responses and development and are key players in reactive oxygen species (ROS) signalling and in innate immunity. In Arabidopsis, the transmission of ROS and pathogen signalling by MAPKs involves the coordinated activation of MPK6 and MPK3; however, the specificity of their negative regulation by phosphatases is not fully known. Here, we present genetic analyses showing that MAPK phosphatase 2 (MKP2) regulates oxidative stress and pathogen defence responses and functionally interacts with MPK3 and MPK6. We show that plants lacking a functional MKP2 gene exhibit delayed wilting symptoms in response to Ralstonia solanacearum and, by contrast, acceleration of disease progression during Botrytis cinerea infection, suggesting that this phosphatase plays differential functions in biotrophic versus necrotrophic pathogen-induced responses. MKP2 function appears to be linked to MPK3 and MPK6 regulation, as indicated by BiFC experiments showing that MKP2 associates with MPK3 and MPK6 in vivo and that in response to fungal elicitors MKP2 exerts differential affinity versus both kinases. We also found that MKP2 interacts with MPK6 in HR-like responses triggered by fungal elicitors, suggesting that MPK3 and MPK6 are subject to differential regulation by MKP2 in this process. We propose that MKP2 is a key regulator of MPK3 and MPK6 networks controlling both abiotic and specific pathogen responses in plants.

Maize AKINbetagamma dimerizes through the KIS/CBM domain and assembles into SnRK1 complexes.

The SNF1/AMPK/SnRK1 complex is an intracellular energy sensor composed of three types of subunits: the SnRK1 kinase and two regulatory, non-catalytic subunits (designated beta and gamma). We have previously described an atypical plant gamma-subunit, AKINbetagamma, which contains an N-terminal tail similar to the so-called KIS domain normally present in beta-subunits. However, it is not known whether AKINbetagamma normally associates with endogenous SnRK1 complexes in vivo, nor how its unique domain structure might contribute to SnRK1 function. Here, we present evidence that maize AKINbetagamma is an integral component of active SnRK1 complexes in plant cells. Using complementary methodological approaches, we also show that AKINbetagamma associates through homomeric interactions mediated by both, the gamma- and, unexpectedly, the KIS/CBM domain.

Overexpression of wheat dehydrin DHN-5 enhances tolerance to salt and osmotic stress in Arabidopsis thaliana.

Late Embryogenesis Abundant (LEA) proteins are associated with tolerance to water-related stress. A wheat (Triticum durum) group 2 LEA proteins, known also as dehydrin (DHN-5), has been previously shown to be induced by salt and abscisic acid (ABA). In this report, we analyze the effect of ectopic expression of Dhn-5 cDNA in Arabidopsis thaliana plants and their response to salt and osmotic stress. When compared to wild type plants, the Dhn-5 transgenic plants exhibited stronger growth under high concentrations of NaCl or under water deprivation, and showed a faster recovery from mannitol treatment. Leaf area and seed germination rate decreased much more in wild type than in transgenic plants subjected to salt stress. Moreover, the water potential was more negative in transgenic than in wild type plants. In addition, the transgenic plants have higher proline contents and lower water loss rate under water stress. Also, Na(+) and K(+) accumulate to higher contents in the leaves of the transgenic plants. Our data strongly support the hypothesis that Dhn-5, by its protective role, contributes to an improved tolerance to salt and drought stress through osmotic adjustment.

Maize DBF1-interactor protein 1 containing an R3H domain is a potential regulator of DBF1 activity in stress responses.

The maize dehydration-responsive element (DRE)-binding factor, DBF1, is a member of the Apetala 2/Ethylene Response Factor transcription factors family and is involved in the regulation of the ABA-responsive gene rab17 through the DRE in an ABA-dependent pathway. In this study we analysed the functionality of DBF1 in abiotic stress responses and found that Arabidopsis plants over-expressing DBF1 were more tolerant to osmotic stress than control plants. In yeast two-hybrid analyses, DBF1 interacted with DBF1-interactor protein 1 (DIP1), a protein containing a conserved R3H single-strand DNA-binding domain. Subcellular localization of DIP1 showed that the protein fusion DIP1-Red Flourescent Protein (RFP) was mainly localized in the cytoplasm. However, after co-transformation of DBF1-GFP and DIP1-RFP, both proteins co-localized in the nucleus. Interestingly, when the N-terminal DBF1-GFP was co-expressed with DIP1-RFP, both proteins co-localized predominantly in the cytoplasmic speckles observed for N-terminal DBF1-GFP fusion protein. These results clearly show in vivo interaction of DBF1 with DIP1 in the cell and that this interaction is necessary for the nuclear localization of DIP1 protein. Analysis of the regulatory effect of the DBF1 and DIP1 interaction on the maize rab17 promoter activity indicated that co-transfection of DBF1 with DIP1 enhances promoter activity in the absence of ABA treatment. We suggest that the regulated association of DBF1 and DIP1 may control the levels of target gene expression during stress conditions.

Isolation and functional characterisation of two new bZIP maize regulators of the ABA responsive gene rab28.

The plant hormone abscisic acid regulates gene expression in response to growth stimuli and abiotic stress. Previous studies have implicated members of the bZIP family of transcription factors as mediators of abscisic acid dependent gene expression through the ABRE cis-element. Here, we identify two new maize bZIP transcription factors, EmBP-2 and ZmBZ-1 related to EmBP-1 and OsBZ-8 families. They are differentially expressed during embryo development; EmBP-2 is constitutive, whereas ZmBZ-1 is abscisic acid-inducible and accumulates during late embryogenesis. Both factors are nuclear proteins that bind to ABREs and activate transcription of the abscisic acid-inducible gene rab28 from maize. EmBP-2 and ZmBZ-1 are phosphorylated by protein kinase CK2 and phosphorylation alters their DNA binding properties. Our data suggest that EmBP-2 and ZmBZ-1 are involved in the expression of abscisic acid inducible genes such as rab28 and their activity is modulated by ABA and by phosphorylation.

A novel higher plant protein tyrosine phosphatase interacts with SNF1-related protein kinases via a KIS (kinase interaction sequence) domain.

A novel protein phosphatase in Arabidopsis thaliana was identified by database searching. This protein, designated AtPTPKIS1, contains a protein tyrosine phosphatase (PTP) catalytic domain and a kinase interaction sequence (KIS) domain. It is predicted to interact with plant SNF1-related kinases (SnRKs), representing central regulators of metabolic and stress responses. AtPTPKIS1 has close homologues in other plant species, both dicots and monocots, but is not found in other kingdoms. The tomato homologue of AtPTPKIS1 was expressed as a recombinant protein and shown to hydrolyse a generic phosphatase substrate, and phosphotyrosine residues in synthetic peptides. The KIS domain of AtPTPKIS1 was shown to interact with the plant SnRK AKIN11 both in vivo in the yeast two-hybrid system, and in vitro in a GST-fusion 'pull down' assay. The genomes of Arabidopsis and other plants contain further predicted proteins related to AtPTPKIS1, which could also interact with SnRKs and act in novel regulatory and signalling pathways.

Arabidopsis thaliana atrab28: a nuclear targeted protein related to germination and toxic cation tolerance.

The Arabidopsis gene Atrab28 has been shown to be expressed during late embryogenesis. The pattern of expression of Atrab28 mRNA and protein during embryo development is largely restricted to provascular tissues of mature embryos, and in contrast to the maize Rab28 homologue it cannot be induced by ABA and dehydration in vegetative tissues. Here, we have studied the subcellular location of Atrab28 protein and the effect of its over-expression in transgenic Arabidopsis plants. The Atrab28 protein was mainly detected in the nucleus and nucleolus of cells from mature embryos. In frame fusion of Atrab28 to the reporter green fluorescent protein (GFP) directed the GFP to the nucleus in transgenic Arabidopsis and in transiently transformed onion cells. Analysis of chimeric constructs identified an N-terminal region of 60 amino acids containing a five amino acid motif QPKRP that was necessary for targeting GFP to the nucleus. These results indicate that Atrab28 protein is targeted to the nuclear compartments by a new nuclear localization signal (NLS). Transgenic Arabidopsis plants, with gain of Atrab28 function, showed faster germination rates under either standard or salt and osmotic stress conditions. Moreover, improved cation toxicity tolerance was also observed not only during germination but also in seedlings. These results suggest a role of Atrab28 in the ion cell balance during late embryogenesis and germination.