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Mitogen-activated protein kinases (MPKs) play roles as critical signal components in the environmental stress responses and developmental processes in plants. Calcium ion (Ca2+) is one of the most essential ubiquitous intracellular second messengers involved in many signal transduction pathways in plants. It was previously known that MPKs are activated by the increasing Ca2+ concentration. However, the mechanism of how Ca2+ activates MPKs is not elucidated yet. In this study, we revealed that Ca2+ could activate MPK signaling pathway via inhibiting the activity of a dual-specificity protein phosphatase1 (DsPTP1) by Ca2+/calmodulin (CaM). We showed that DsPTP1 directly interacts with MPK6 in vitro and in vivo. DsPTP1 was able to inactivate the active MPK6 by dephosphorylation. Interestingly, the DsPTP1-mediated dephosphorylation of MPK6 was strongly inhibited by Ca2+/CaM. Moreover, this inhibition was caused by the binding of CaM to the calmodulin-binding domain II (CaMBDII) of DsPTP1. This study implies that Ca2+/CaM is involved in the activation of MPKs through the inhibition of DsPTP1.

Post-translational modification of proteins regulates signaling cascades in eukaryotic system, including plants. Among these modifications, phosphorylation plays an important role in modulating the functional properties of proteins. Plants perceive environmental cues that directly affect the phosphorylation status of many target proteins. To determine the effect of environmentally induced phosphorylation in plants, in vivo methods must be developed. Various in vitro methods are available but, unlike in animals, there is no optimized methodology for detecting protein phosphorylation in planta. Therefore, in this study, a robust, and easy to handle Phos-Tag Mobility Shift Assay (PTMSA) is developed for the in vivo detection of protein phosphorylation in plants by empirical optimization of methods previously developed for animals. Initially, the detection of the phosphorylation status of target proteins using protocols directly adapted from animals failed. Therefore, we optimized the steps in the protocol, from protein migration to the transfer of proteins to PVDF membrane. Supplementing the electrophoresis running buffer with 5 mM NaHSO3 solved most of the problems in protein migration and transfer. The optimization of a fast and robust protocol that efficiently detects the phosphorylation status of plant proteins was successful. This protocol will be a valuable tool for plant scientists interested in the study of protein phosphorylation.

Flavonoids are well known for the coloration of plant organs to protect UV and ROS and to attract pollinators as well. Flavonoids also play roles in many aspects of physiological processes including pathogen resistance. However, the molecular mechanism to explain how flavonoids play roles in pathogen resistance was not extensively studied. In this study, we investigated how naringenin, the first intermediate molecule of the flavonoid biosynthesis, functions as an activator of pathogen resistances. The transcript levels of two pathogenesis-related (PR) genes were increased by the treatment with naringenin in Arabidopsis. Interestingly, we found that naringenin triggers the monomerization and nuclear translocation of non-expressor of pathogenesis-related genes 1 (NPR1) that is a transcriptional coactivator of PR gene expression. Naringenin can induce the accumulation of salicylic acid (SA) that is required for the monomerization of NPR1. Furthermore, naringenin activates MPK6 and MPK3 in ROS-dependent, but SA-independent manners. By using a MEK inhibitor, we showed that the activation of a MAPK cascade by naringenin is also required for the monomerization of NPR1. These results suggest that the pathogen resistance by naringenin is mediated by the MAPK- and SA-dependent activation of NPR1 in Arabidopsis.

The ethylene-responsive element binding factor (ERF) family is a large family of transcription factors involved in plant development and environmental stress responses. We previously reported the identification of 29 putative substrates of Mitogen-activated Protein Kinase3 (AtMPK3), AtMPK4 and AtMPK6, based on a solid-phase phosphorylation screening using a lambda phage expression library in Arabidopsis thaliana. In this study, a putative MPK substrate, AtERF72 (At3g16770), was strongly phosphorylated by AtMPK6 on the serine residue at position 151 (Ser151). AtERF72 binds to the GCC box (AGCCGCC) in the promoters of several pathogenesis-related (PR) genes and activates their transcription. We also show that the DNA-binding activity of AtERF72 is enhanced upon phosphorylation by AtMPK6 in vitro. In addition, transient co-expression experiments in Arabidopsis protoplasts revealed that effector constructs expressing a mutant variant of AtERF72, AtERF72S151D (carrying a Ser to aspartic acid [Asp] substitution at amino acid position 151) showed higher expression of the β-glucuronidase (GUS) reporter gene driven by the GCC box element than effector constructs expressing the wild-type AtERF72. Furthermore, yeast two-hybrid assays revealed that the interaction between AtERF72S151D and TGA4/OBF4 was stronger than that between wild-type AtERF72 and TGA4/OBF4. Since AtERF72S151D is equivalent to AtERF72 phosphorylated by AtMPK6 at Ser151, these results suggest that the phosphorylation of AtERF72 by AtMPK6 triggers an event of transcriptional regulation from defence signalling in Arabidopsis.

Lateral root development is known to be regulated by Aux/IAA-ARF modules in Arabidopsis thaliana. As components, several Aux/IAAs have participated in these Aux/IAA-ARF modules. In this study, to identify the biological function of IAA15 in plant developments, transgenic plant overexpressing the gain-of-function mutant of IAA15 (IAA15P75S OX) under the control of dexamethasone (DEX) inducible promoter, in which IAA15 protein was mutated by changing Pro-75 residue to Ser at the degron motif in conserved domain II, was constructed. As a result, we found that IAA15P75S OX plants show a decreased number of lateral roots. Coincidently, IAA15 promoter-GUS reporter analysis revealed that IAA15 transcripts were highly detected in all stages of developing lateral root tissues. It was also verified that the IAA15P75S protein is strongly stabilized against proteasome-mediated protein degradation by inhibiting its poly-ubiquitination, resulting in the transcriptional repression of auxin-responsive genes. In particular, transcript levels of LBD16 and LBD29, which are positive regulators of lateral root formation, dramatically repressed in IAA15P75S OX plants. Furthermore, it was elucidated that IAA15 interacts with ARF7 and ARF19 and binds to the promoters of LBD16 and LBD29, strongly suggesting that IAA15 represses lateral root formation through the transcriptional suppression of LBD16 and LBD29 by inhibiting ARF7 and ARF19 activity. Taken together, this study suggests that IAA15 also plays a key negative role in lateral root formation as a component of Aux/IAA-ARF modules.

Seed germination is a complex biological process controlled by various regulators, including phytohormones. Among these, abscisic acid and gibberellic acid inhibit and promote seed germination, respectively. Many studies have addressed the biological roles of auxin in plant growth and development, but very few have considered its role in seed germination. Here, we identified a novel function of the auxin signaling repressor Aux/IAA8 during seed germination. The IAA8 loss-of-function mutant iaa8-1 exhibited delayed seed germination. The phenotype of iaa8-1 was restored by ectopic expression of IAA8. Interestingly, IAA8 accumulated to high levels during seed germination, which was achieved not only by increased protein synthesis but also by the stabilization of IAA8 protein. We also showed that IAA8 down-regulates the transcription of ABSCISIC ACID INSENSITIVE3 (ABI3), a negative regulator of seed germination. Our study, thus strongly suggest that the auxin signaling repressor IAA8 acts as a positive regulator of seed germination in Arabidopsis thaliana.

Sessile plants reprogram their metabolic and developmental processes during adaptation to prolonged environmental stresses. To understand the molecular mechanisms underlying adaptation of plant cells to saline stress, we established callus suspension cell cultures from Arabidopsis roots adapted to high salt for an extended period of time. Adapted cells exhibit enhanced salt tolerance compared with control cells. Moreover, acquired salt tolerance is maintained even after the stress is relieved, indicating the existence of a memory of acquired salt tolerance during mitotic cell divisions, known as mitotic stress memory. Metabolite profiling using 1H-nuclear magnetic resonance (NMR) spectroscopy revealed metabolic discrimination between control, salt-adapted and stress-memory cells. Compared with control cells, salt-adapted cells accumulated higher levels of sugars, amino acids and intermediary metabolites in the shikimate pathway, such as coniferin. Moreover, adapted cells acquired thicker cell walls with higher lignin contents, suggesting the importance of adjustments of physical properties during adaptation to elevated saline conditions. When stress-memory cells were reverted to normal growth conditions, the levels of metabolites again readjusted. Whereas most of the metabolic changes reverted to levels intermediate between salt-adapted and control cells, the amounts of sugars, alanine, γ-aminobutyric acid and acetate further increased in stress-memory cells, supporting a view of their roles in mitotic stress memory. Our results provide insights into the metabolic adjustment of plant root cells during adaptation to saline conditions as well as pointing to the function of mitotic memory in acquired salt tolerance.

Phytocystatins (PhyCYSs) are plant-specific proteinaceous inhibitors that are implicated in protein turnover and stress responses. Here, we characterized a PhyCYS from Arabidopsis thaliana, which was designated AtCYS5. RT-qPCR analysis showed that the expression of AtCYS5 in germinating seeds was induced by heat stress (HS) and exogenous abscisic acid (ABA) treatment. Analysis of the expression of the β-glucuronidase reporter gene under the control of the AtCYS5 promoter showed that AtCYS5 expression during seed germination was induced by HS and ABA. Constitutive overexpression of AtCYS5 driven by the cauliflower mosaic virus 35S promoter led to enhanced HS tolerance in transgenic Arabidopsis, which was characterized by higher fresh weight and root length compared to wild-type (WT) and knockout (cys5) plants grown under HS conditions. The HS tolerance of At-CYS5-overexpressing transgenic plants was associated with increased insensitivity to exogenous ABA during both seed germination and post-germination compared to WT and cys5. Although no HS elements were identified in the 5'-flanking region of AtCYS5, canonical ABA-responsive elements (ABREs) were detected. AtCYS5 was upregulated in ABA-treated protoplasts transiently co-expressing this gene and genes encoding bZIP ABRE-binding factors (ABFs and AREB3). In the absence of ABA, ABF1 and ABF3 directly bound to the ABREs in the AtCYS5 promoter, which activated the transcription of this gene in the presence of ABA. These results suggest that an ABA-dependent pathway plays a positive role in the HS-responsive expression of AtCYS5 during seed germination and post-germination growth.

Phosphorylation of substrate proteins by mitogen-activated protein kinases (MPKs) determines the specific cellular responses elicited by a particular extracellular stimulus. However, downstream targets of plant MPKs remain poorly characterized. In this study, 29 putative substrates of AtMPK3, AtMPK4 and AtMPK6 were identified by solid-phase phosphorylation screening of a λ phage expression library constructed from combined mRNAs from salt-treated, pathogen-treated and mechanically wounded Arabidopsis seedlings. To test the efficiency of this screening, we performed in vitro kinase assay with 10 recombinant fusion proteins. All proteins were phosphorylated by AtMPK3, AtMPK4 and AtMPK6, indicating the efficiency of this screening procedure. To confirm phosphorylation of isolated substrates by plant MPKs, we performed in-gel kinase assays. All test substrates were strongly phosphorylated by wounding or H2O2-activated AtMPK3 and AtMPK6. Three substrates, encoded by genes At2g41430, At2g41900, and At3g16770, were strongly phosphorylated, suggesting a function as AtMPK substrates. The type of screening provides a powerful way for identifying potential substrates of MAP kinases responsive to biotic and abiotic stresses.

Heat shock factors (Hsfs) are central regulators of abiotic stress responses, especially heat stress responses, in plants. In the current study, we characterized the activity of the Hsf gene HsfA3 in Arabidopsis under oxidative stress conditions. HsfA3 transcription in seedlings was induced by reactive oxygen species (ROS), exogenous hydrogen peroxide (H2O2), and an endogenous H2O2 propagator, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). HsfA3-overexpressing transgenic plants exhibited increased oxidative stress tolerance compared to untransformed wild-type plants (WT), as revealed by changes in fresh weight, chlorophyll fluorescence, and ion leakage under light conditions. The expression of several genes encoding galactinol synthase (GolS), a key enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs), which function as antioxidants in plant cells, was induced in HsfA3 overexpressors. In addition, galactinol levels were higher in HsfA3 overexpressors than in WT under unstressed conditions. In transient transactivation assays using Arabidopsis leaf protoplasts, HsfA3 activated the transcription of a reporter gene driven by the GolS1 or GolS2 promoter. Electrophoretic mobility shift assays showed that GolS1 and GolS2 are directly regulated by HsfA3. Taken together, these findings provide evidence that GolS1 and GolS2 are directly regulated by HsfA3 and that GolS enzymes play an important role in improving oxidative stress tolerance by increasing galactinol biosynthesis in Arabidopsis.

Osmotic stress is induced by several environmental stresses such as drought, cold and salinity. Osmotic stress may finally leads to oxidative damage, ionic imbalance and growth inhibition in plants. Gene expression analyses indicated that ZAT10 transcription factor, a novel substrate of Arabidopsis MPKs, could be induced by environmental stresses that result in osmotic stress. As previously reported, ZAT10 overexpressing transgenic plants showed enhanced tolerance to osmotic stress. However, in contrast to previous report, a zat10 knockout mutant showed an osmotic stress sensitive phenotype. To determine the biological function of EAR domain and phosphorylation sites of ZAT10, we constructed two transgenic plants expressing two ZAT10 mutant proteins having EAR domain mutations (ZAT10EAR) and phosphorylation site mutations (ZAT10AA). The phenotype of zat10 was complemented by the expression of ZAT10EAR mutant, however not by the expression of ZAT10AA mutant, indicating that the phosphorylation sites in ZAT10 by MPKs are involved in stress tolerance but the EAR domain is not. In this report, we suggest that ZAT10 function as a positive regulator in osmotic stress tolerance and the phosphorylation of ZAT10 is required for its function in Arabidopsis.

The expression of CBF (C-repeat-binding factor) genes is required for freezing tolerance in Arabidopsis thaliana. CBFs are positively regulated by INDUCER OF CBF EXPRESSION1 (ICE1) and negatively regulated by MYB15. These transcription factors directly interact with specific elements in the CBF promoters. Mitogen-activated protein kinase (MAPK/MPK) cascades function upstream to regulate CBFs. However, the mechanism by which MPKs control CBF expression during cold stress signaling remains unknown. This study showed that the activity of MYB15, a transcriptional repressor of cold signaling, is regulated by MPK6-mediated phosphorylation. MYB15 specifically interacts with MPK6, and MPK6 phosphorylates MYB15 on Ser168. MPK6-induced phosphorylation reduced the affinity of MYB15 binding to the CBF3 promoter and mutation of its phosphorylation site (MYB15S168A) enhanced the transcriptional repression of CBF3 by MYB15. Furthermore, transgenic plants overexpressing MYB15S168A showed significantly reduced CBF transcript levels in response to cold stress, compared with plants overexpressing MYB15. The MYB15S168A-overexpressing plants were also more sensitive to freezing than MYB15-overexpressing plants. These results suggest that MPK6-mediated regulation of MYB15 plays an important role in cold stress signaling in Arabidopsis.

Nitric oxide (NO) is known for its role in the activation of plant defense responses. To examine the involvement and mode of action of NO in plant defense responses, we introduced calmodulin-dependent mammalian neuronal nitric oxide synthase (nNOS), which controls the CaMV35S promoter, into wild-type and NahG tobacco plants. Constitutive expression of nNOS led to NO production and triggered spontaneous induction of leaf lesions. Transgenic plants accumulated high amounts of H(2)O(2), with catalase activity lower than that in the wild type. nNOS transgenic plants contained high levels of salicylic acid (SA), and they induced an array of SA-, jasmonic acid (JA)-, and/or ethylene (ET)-related genes. Consequently, NahG co-expression blocked the induction of systemic acquired resistance (SAR)-associated genes in transgenic plants, implying SA is involved in NO-mediated induction of SAR genes. The transgenic plants exhibited enhanced resistance to a spectrum of pathogens, including bacteria, fungi, and viruses. Our results suggest a highly ranked regulatory role for NO in SA-, JA-, and/or ET-dependent pathways that lead to disease resistance.

Calmodulin (CaM), a key Ca2+ sensor, regulates diverse cellular processes by modulating the activity of a variety of enzymes and proteins. However, little is known about the biological function of CaM in plant development. In this study, an ASYMMETRIC LEAVES1 (AS1) transcription factor was isolated as a CaM-binding protein. AS1 contains two putative CaM-binding domains (CaMBDs) at the N-terminus. Using domain mapping analysis, both predicted domains were identified as authentic Ca2+ -dependent CaMBDs. We identified three hydrophobic amino acid residues for CaM binding, Trp49 in CaMBDI, and Trp81 and Phe103 in CaMBDII. The interactions of AS1 with CaM were verified in yeast and plant cells. Based on electrophoretic mobility shift assays, CaM inhibited the DNA-binding activity of the AS1/AS2 complex to two cis-regulatory motifs in the KNAT1 promoter. Furthermore, CaM relieved the suppression of KNAT1 transcription by AS1 not only in transient expression assays of protoplasts but also by the overexpression of a CaM-binding negative form of AS1 in as1 mutant plant. Our study suggests that CaM, a calcium sensor, can be involved in the transcriptional control of meristem cell-specific genes by the inhibition of AS1 under the condition of higher levels of Ca2+ in plants.

Plants have developed various regulatory pathways to adapt to environmental stresses. In this study, we identified Arabidopsis MKKK20 as a regulator in the response to osmotic stress. mkkk20 mutants were found to be sensitive to high concentration of salt and showed higher water loss rates than wild-type (WT) plants under dehydration conditions. In addition, mkkk20 mutants showed higher accumulation of superoxide, a reactive oxygen species (ROS), compared to WT plants under high salt condition. In contrast, transgenic plants overexpressing MKKK20 displayed tolerance to salt stress. MKKK20 transcripts were increased by the treatments with NaCl, mannitol, MV, sorbitol, and cold, suggesting that MKKK20 is involved in the response to osmotic, ROS, and cold stresses. In-gel kinase assay showed that MKKK20 regulates the activity of MPK6 under NaCl, cold, and H(2)O(2) treatments. Taken together, our results suggest that MKKK20 might be involved in the response to various abiotic stresses, especially osmotic stress, through its regulation of MPK6 activity.

Calcium () signals have been implicated in regulating plant development and responses to the environmental stresses including a programmed cell death pathway. In animals and plants, cytosolic signals have been involved in the activation of programmed cell death (PCD). Recently, we reported that disruption of Arabidopsis vacuolar utoinhibited -TPases (ACAs), ACA4 and ACA11, resulted in the activation of a salicylic acid-dependent programmed cell death pathway. Although extensive studies have revealed various components of a PCD in plants, executors to directly induce PCD are well unknown. Here, we provide that the vacuolar processing enzymes (VPEs) are involved in a PCD induced by the double knockout mutant of vacuolar -ATPases in Arabidopsis. The gene expression of VPE was rapidly up-regulated and the enzyme activity of VPE was increased in the double mutant plants. We also generated aca4/aca11/avpe, aca4/aca11/vpe and aca4/aca11/avpe/vpe mutant plants. Although cell death phenotype of the double mutant plants was not completely disappeared in the triple and quadruple mutant plants, the triple and quadruple mutant plants showed to significantly delay cell death phenotype of the double mutant plants. These results suggest that the VPE is involved in the HR-like cell death in the double mutant of vacuolar -ATPases in Arabidopsis.

Calmodulin (CaM), a Ca2+ binding protein in eukaryotes, mediates cellular Ca2+ signals in response to a variety of biotic and abiotic external stimuli. The Ca2+-bound CaM transduces signals by modulating the activities of numerous CaM-binding proteins. As a CaM binding protein, AtCBP63 (Arabidopsis thaliana CaM-binding protein 63 kD) has been known to be positively involved in plant defense signaling pathway. To investigate the pathogen resistance function of AtCBP63 in potato, we constructed transgenic potato (Solanum tuberosum L.) plants constitutively overexpressing AtCBP63 under the control of cauliflower mosaic virus (CaMV) 35S promoter. The overexpression of the AtCBP63 in potato plants resulted in the high level induction of pathogenesis-related (PR) genes such as PR-2, PR-3 and PR-5. In addition, the AtCBP63 transgenic potato showed significantly enhanced resistance against a pathogen causing bacterial soft rot, Erwinia carotovora ssp. Carotovora (ECC). These results suggest that a CaM binding protein from Arabidopsis, AtCBP63, plays a positive role in pathogen resistance in potato.

The dehydration responsive element binding protein 2C (DREB2C) is a dehydration responsive element/C-repeat (DRE/CRT)-motif binding transcription factor that induced by mild heat stress. Previous experiments established that overexpression of DREB2C cDNA driven by the cauliflower mosaic virus 35S promoter (35S:DREB2C) resulted in increased heat tolerance in Arabidopsis. We first analyzed the proteomic profiles in wild-type and 35S:DREB2C plants at a normal temperature (22 degrees C), but could not detect any differences between the proteomes of wild-type and 35S:DREB2C plants. The transcript level of DREB2C in 35S:DREB2C plants after treatment with mild heat stress was increased more than two times compared with expression in 35S:DREB2C plants under unstressed condition. A proteomic approach was used to decipher the molecular mechanisms underlying thermotolerance in 35S:DREB2C Arabidopsis plants. Eleven protein spots were identified as being differentially regulated in 35S:DREB2C plants. Moreover, in silico motif analysis showed that peptidyl-prolyl isomerase ROC4, glutathione transferase 8, pyridoxal biosynthesis protein PDX1, and elongation factor Tu contained one or more DRE/CRT motifs. To our knowledge, this study is the first to identify possible targets of DREB2C transcription factors at the protein level. The proteomic results were in agreement with transcriptional data.

The transcription of soybean (Glycine max) calmodulin isoform-4 (GmCaM-4) is dramatically induced within 0.5 h of exposure to pathogen or NaCl. Core cis-acting elements that regulate the expression of the GmCaM-4 gene in response to pathogen and salt stress were previously identified, between -1,207 and -1,128 bp, and between -858 and -728 bp, in the GmCaM-4 promoter. Here, we characterized the properties of the DNA-binding complexes that form at the two core cis-acting elements of the GmCaM-4 promoter in pathogen-treated nuclear extracts. We generated GUS reporter constructs harboring various deletions of approximately 1.3-kb GmCaM-4 promoter, and analyzed GUS expression in tobacco plants transformed with these constructs. The GUS expression analysis suggested that the two previously identified core regions are involved in inducing GmCaM-4 expression in the heterologous system. Finally, a transient expression assay of Arabidopsis protoplasts showed that the GmCaM-4 promoter produced greater levels of GUS activity than did the CaMV35S promoter after pathogen or NaCl treatments, suggesting that the GmCaM-4 promoter may be useful in the production of conditional gene expression systems.

Calcium ion () is thought to play the important role as a second messenger for signal transduction that results in various physiological responses to cope with developmental programs and environmental changes in plant. In plant cells, the central vacuole functions as a major calcium store, which is important for both signal transduction and preventing cytotoxicity. Although there is evidence for the biochemical characterizations of a calmodulin-regulated -ATPase (ACA11) localized to vacuole membrane, the biological function to ACA11 in plant has not been verified. In this study, we show that the cell death as the hypersensitive response (HR) in mature leaves is induced in transgenic plant of a vacuole ACA-type -ATPase, ACA11. Evidence that cell death phenotype is the result of ACA11 gene silencing is provided by Western blot assay using membrane fraction proteins extracted from transgenic plant. The 3, 3`-diaminobenzidine (DAB) staining study provides that the cell death is caused by the increase of reactive oxygen species (ROS) in mature leaves of transgenic plants.

Voltage-dependent anion channels (VDACs) are reported to be porin-type, beta-barrel diffusion pores. They are prominently localized in the outer mitochondrial membrane and are involved in metabolite exchange between the organelle and the cytosol. In this study, we have investigated a family of VDAC isoforms in Arabidopsis thaliana (AtVDAC). We have shown that the heterologous expression of AtVDAC proteins can functionally complement a yeast mutant lacking the endogenous mitochondrial VDAC gene. AtVDACs tagged with GFP were localized to mitochondria in both yeast and plant cells. We also looked at the response of AtVDACs to biotic and abiotic stresses and found that four AtVDAC transcripts were rapidly up-regulated in response to a bacterial pathogen.

Sessile plants have developed a very delicate system to sense diverse kinds of endogenous developmental cues and exogenous environmental stimuli by using a simple Ca2+ ion. Calmodulin (CaM) is the predominant Ca2+ sensor and plays a crucial role in decoding the Ca2+ signatures into proper cellular responses in various cellular compartments in eukaryotes. A growing body of evidence points to the importance of Ca2+ and CaM in the regulation of the transcriptional process during plant responses to endogenous and exogenous stimuli. Here, we review recent progress in the identification of transcriptional regulators modulated by Ca2+ and CaM and in the assessment of their functional significance during plant signal transduction in response to biotic and abiotic stresses and developmental cues.

Calmodulin-regulated protein phosphorylation plays a pivotal role in amplifying and diversifying the action of calcium ion. In this study, we identified a calmodulin-binding receptor-like protein kinase (CBRLK1) that was classified into an S-locus RLK family. The plasma membrane localization was determined by the localization of CBRLK1 tagged with a green fluorescence protein. Calmodulin bound specifically to a Ca2+-dependent calmodulin binding domain in the C-terminus of CBRLK1. The bacterially expressed CBRLK1 kinase domain could autophosphorylate and phosphorylates general kinase substrates, such as myelin basic proteins. The autophosphorylation sites of CBRLK1 were identified by mass spectrometric analysis of phosphopeptides. Structured summary MINT- 6800947 :CBRLK1 (uniprotkb: Q9ZT06 ) and AtCaM2 (uniprotkb: P25069 ) bind (MI: 0407 ) by electrophoretic mobility shift assay (MI: 0413 ) MINT- 6800966 :AtCaM2 (uniprotkb: P25069 ) and CBRLK1 (uniprotkb: Q9ZT06 ) bind (MI: 0407 ) by competition binding (MI: 0405 ) MINT- 6800930 :CBRLK1 (uniprotkb: Q9ZT06 ) binds (MI: 0407 ) to AtCaM2 (uniprotkb: P25069 ) by far Western blotting (MI: 0047 ) MINT- 6800978 :AtCaM2 (uniprotkb: P25069 ) physically interacts (MI: 0218 ) with CBRLK1 (uniprotkb: Q9ZT06 ) by cytoplasmic complementation assay (MI: 0228 )

The mitogen-activated protein kinases (MAPKs) are key signal transduction molecules, which respond to various external stimuli. The MAPK phosphatases (MKPs) are known to be negative regulators of MAPKs in eukaryotes. We screened an Arabidopsis cDNA library using horseradish peroxidase-conjugated calmodulin (CaM), and isolated AtMKP1 as a CaM-binding protein. Recently, tobacco NtMKP1 and rice OsMKP1, two orthologs of Arabidopsis AtMKP1, were reported to bind CaM via a single putative CaM binding domain (CaMBD). However, little is known about the regulation of phosphatase activity of plant MKP1s by CaM binding. In this study, we identified two Ca2+-dependent CaMBDs within AtMKP1. Specific binding of CaM to two different CaMBDs was verified using a gel mobility shift assay, a competition assay with a Ca2+/CaM-dependent enzyme, and a split-ubiquitin assay. The peptides for two CaMBDs, CaMBDI and CaMBDII, bound CaM in a Ca2+-dependent manner, and the binding affinity of CaMBDII was found to be higher than that of CaMBDI. CaM overlay assays using mutated CaMBDs showed that four amino acids, Trp453 and Leu456 in CaMBDI and Trp678 and Ile684 in CaMBDII, play a pivotal role in CaM binding. Moreover, the phosphatase activity of AtMKP1 was increased by CaM in a Ca2+-dependent manner. Our results suggest that two important signaling pathways, Ca2+ signaling and the MAPK signaling cascade, are connected in plants via the regulation of AtMKP1 activity. To our knowledge, this is the first report to show that the biochemical activity of MKP1 in plants is regulated by CaM.

Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates diverse cellular functions by modulating the activity of a variety of proteins. However, little is known about how CaM directly regulates transcription. Screening of an Arabidopsis cDNA expression library using horseradish peroxidase-conjugated calmodulin as a probe identified a calmodulin-binding NAC protein (CBNAC). Using gel overlay assays, a Ca2+-dependent CaM-binding domain was identified in the C terminus of this protein. Specific binding of CaM to CaM-binding domain was corroborated by site-directed mutagenesis and a split-ubiquitin assay. Using a PCR-mediated random binding site selection method, we identified a DNA-binding sequence (CBNACBS) for CBNAC, which consisted of a GCTT core sequence flanked on both sides by other frequently repeating sequences (TTGCTTANNNNNNAAG). CBNAC was able to bind to CBNACBS, which resulted in the repression of transcription in Arabidopsis protoplasts. Interestingly, the transcriptional repression mediated by CBNAC was enhanced by CaM. These results suggest that CBNAC may be a CaM-regulated transcriptional repressor in Arabidopsis.

The dehydration responsive element binding protein 2 (DREB2) subgroup belongs to the plant-specific APETALA2/ethylene-responsive element binding factor (AP2/ERF) family of transcription factors. We have characterized cDNA encoding Arabidopsis thaliana DREB2C, which is induced by mild heat stress. Both an electrophoretic mobility shift assay (EMSA) and a yeast one-hybrid assay revealed that DREB2C(145-528) was able to form a complex with the dehydration responsive element/C-repeat (DRE/CRT; A/GCCGAC) motif. A trans-activating ability test in yeast demonstrated that DREB2C could effectively function as a trans-activator. Constitutive expression of DREB2C under the control of the cauliflower mosaic virus (CaMV) 35S promoter led to enhanced thermotolerance in transgenic lines of Arabidopsis. Microarray and RT-PCR analyses of transgenic plants revealed that DREB2C regulates expression of several heat stress-inducible genes that contain DRE/CRT elements in their promoters. From these data, we deduced that DREB2C is a regulator of heat stress tolerance in Arabidopsis.

Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates diverse cellular functions by modulating the activity of a variety CaM-binding proteins (CaMBPs). Because eukaryotes have multiple CaMBPs, it is important to isolate and characterize them in different tissues and conditions. So far a number of CaMBPs have been identified through classical screening methods. Many classes of proteins have been predicted to bind CaMs based on their structural homology with already known targets. In an effort to develop a method for large-scale analysis of CaMBPs in Arabidopsis, we have generated a transgenic plants overexpressing AtCaM2-GFP. We performed protein pull-down assay to test whether exogenously expressed AtCaM2-GFP proteins can interact with CaMBPs. The exogenously expressed AtCaM2-GFP could strongly interact with a CaMBP, AS1 protein. This result suggests that AtCaM2-GFP in transgenic plants may interact with many CaMBPs in plant cell. Therefore, we will be able to isolate kinds of CaMBPs by using these transgenic plants in many different tissue and environments.

In eukaryotes, mitogen-activated protein kinases (MAPKs) play important roles in various developmental processes and in environmental stress responses. Here, we show that alternative splicing of the OsBWMK1, a member of the rice MAPK family, generates three transcript variants, OsBWMK1L, OsBWMK1M, and OsBWMK1S. The OsBWMK1L transcript variant was highly and constitutively expressed in all rice tissues tested and its expression was not altered by various stress conditions, whereas OsBWMK1M and OsBWMK1S were normally expressed at low levels but were induced by various stresses. A transient expression assay demonstrated that OsBWMK1L::GFP and OsBWMK1M::GFP were predominantly localized in the cytoplasm, whereas most OsBWMK1S::GFP was localized in the nucleus. Moreover, treatment with defense signaling related molecules, such as H2O2 and SA, induced translocation of OsBWMK1 isoforms from the cytoplasm to the nucleus. Thus, our results suggest that alternative splicing of OsBWMK1 generates three different transcript variants that produce proteins with different subcellular localizations.

2-Cys peroxiredoxins (Prxs) play important roles in the antioxidative defense systems of plant chloroplasts. In order to determine the interaction partner for these proteins in Arabidopsis, we used a yeast two-hybrid screening procedure with a C175S-mutant of Arabidopsis 2-Cys Prx-A as bait. A cDNA encoding an NADPH-dependent thioredoxin reductase (NTR) isotype C was identified and designated ANTR-C. We demonstrated that this protein effected efficient transfer of electrons from NADPH to the 2-Cys Prxs of chloroplasts. Interaction between 2-Cys Prx-A and ANTR-C was confirmed by a pull-down experiment. ANTR-C contained N-terminal TR and C-terminal Trx domains. It exhibited both TR and Trx activities and co-localized with 2-Cys Prx-A in chloroplasts. These results suggest that ANTR-C functions as an electron donor for plastidial 2-Cys Prxs and represents the NADPH-dependent TR/Trx system in chloroplasts.

If plants are pre-exposed to moderate heat stress, they can acquire enhanced tolerance to otherwise lethal high temperatures. To elucidate gene regulatory events involved in the acquisition of thermotolerance, we here conducted a comprehensive transcriptomic analysis. Chinese cabbage microarrays (Brassica rapa EST 6.4 K) were used to compare gene expression of 7-day old seedlings exposed to permissive high temperatures over a time course (0, 0.25, 1, 3, and 12 h at 37 °C). Expression profiling showed that heat treatment triggered significant accumulation of heat shock proteins over time and that some cell wall-modificatory (CWM) genes were up-regulated at the later time points. This up-regulation of CWM genes was verified by reverse-transcription (RT)-PCR and histochemical analysis also provided evidence that cell wall thickness is increased by permissive heat treatment. These observations imply that the activation of CWM-related genes by permissive high temperature stress is an important response for the acquisition of thermotolerance.

Salt stress is one of major environmental factors influencing plant growth and development. To identify salt tolerance determinants in higher plants, a large-scale screen was conducted with a bialaphos marker-based T-DNA insertional collection of Arabidopsis ecotype C24 mutants. One line for salt stress-sensitive mutant (referred to as ssm1) exhibited increased sensitivity to both ionic (NaCl) and nonionic (mannitol) osmotic stress in a root growth assay. This result suggests that ssm1 mutant is involved in ion homeostasis and osmotic compensation in plant. Molecular cloning of the genomic DNA flanking T-DNA insert of ssm1 mutant was achieved by mutant genomic DNA library screening. T-DNA insertion appeared in the first exon of an open reading frame on F3M18.7, which is the same as AtSYP61. SSM1 is SYP61/OSM1 that is a member of the SNARE superfamily of proteins required for vesicular/target membrane fusions and factor related to abiotic stress.

Calreticulin (CRT) is a ubiquitously expressed, high capacity Ca(2+)-binding protein that is involved in intracellular Ca(2+) homeostasis and molecular chaperoning in the endoplasmic reticulum (ER). A cDNA encoding a calreticulin, BrCRT1 (Brassica rapa Calreticulin 1), has been isolated from Chinese cabbage (B. rapa subsp. pekinensis) flower bud. Constitutive over-expression of the BrCRT1 gene promotes robust shoot production and root formation at sub-optimal concentrations of BA/NAA, which are important factors controlling plant regeneration in tissue culture. In contrast, the suppressed BrCRT1 line exhibited a slight reduction of shoot and root regeneration. In spite of enhanced regeneration in tissue culture, the seedling and plant growth rate was inhibited in soil. The steady state level of BrCRT1 transcripts was sensitive to exogenous auxins and cytokinins, and rapidly accumulated within 30 min, and this induction required de novo protein synthesis. Together with the results of transgenic tobacco plants and mRNA analysis in Chinese cabbage, our data suggest that BrCRT1 genes may up-regulate the competency of vegetative tissue to respond to hormonal signals involved in shoot and root regeneration processes.

Calmodulin (CaM) influences many cellular processes by interacting with various proteins. Here, we isolated AtBAG6, an Arabidopsis CaM-binding protein that contains a central BCL-2-associated athanogene (BAG) domain. In yeast and plants, overexpression of AtBAG6 induced cell death phenotypes consistent with programmed cell death (PCD). Recombinant AtBAG6 had higher affinity for CaM in the absence of free Ca2 + than in its presence. An IQ motif (IQXXXRGXXXR, where X denotes any amino-acid) was required for Ca2 +-independent CaM complex formation and single amino-acid changes within this motif abrogated both AtBAG6-activated CaM-binding and cell death in yeast and plants. A 134-amino-acid stretch, encompassing both the IQ motif and BAG domain, was sufficient to induce cell death. Agents generating oxygen radicals, which are known to be involved in plant PCD, specifically induced the AtBAG6 transcript. Collectively, these results suggest that AtBAG6 is a stress-upregulated CaM-binding protein involved in plant PCD.

Calmodulin (CaM), a key Ca(2+) sensor in eukaryotes, regulates diverse cellular processes by interacting with many proteins. To identify Ca(2+)/CaM-mediated signaling components, we screened an Arabidopsis expression library with horseradish peroxidase-conjugated Arabidopsis calmodulin2 (AtCaM2) and isolated a homolog of the UBP6 deubiquitinating enzyme family (AtUBP6) containing a Ca(2+)-dependent CaM-binding domain (CaMBD). The CaM-binding activity of the AtUBP6 CaMBD was confirmed by CaM mobility shift assay, phosphodiesterase competition assay and site-directed mutagenesis. Furthermore, expression of AtUBP6 restored canavanine resistance to the Deltaubp6 yeast mutant. This is the first demonstration that Ca(2+) signaling via CaM is involved in ubiquitin-mediated protein degradation and/or stabilization in plants.

Vernalization is induced by prolonged cold and accelerates flowering. To monitor the genome-wide transcriptome change that identifies the genes that are differently up-regulated depending on the duration of cold exposure, we fabricated a Chinese cabbage cDNA microarray and used it to identify genes that are up-regulated by short-term (1-week) and long-term (4-week) cold. The overall expression patterns between 1- and 4-week cold treated seedlings were very similar but some genes in the cell growth/division/DNA synthesis functional category were regulated differently. Prolonged cold specifically up-regulated several heat shock proteins ( HSPs ), and small HSPs were more significant. Among the up-regulated sHSPs , we chose the small HSP 17.6-II and confirmed its up-regulation in response to long- but not short-term cold exposure by Northern analysis. And its constitutive overexpression moderately accelerated flowering.

Calmodulin (CaM) is a ubiquitous Ca2+-binding protein known to regulate diverse cellular functions by modulating the activity of various target proteins. We isolated a cDNA encoding AtWRKY7, a novel CaM-binding transcription factor, from an Arabidopsis expression library with horseradish peroxidase-conjugated CaM. CaM binds specifically to the Ca2+-dependent CaM-binding domain (CaMBD) of AtWRKY7, as shown by site-directed mutagenesis, a gel mobility shift assay, a split-ubiquitin assay, and a competition assay using a Ca2+/CaM-dependent enzyme. Furthermore, we show that the CaMBD of AtWRKY7 is a conserved structural motif (C-motif) found in group IId of the WRKY protein family.

Rapid, large-scale generation of a Ds transposant population was achieved using a regeneration procedure involving tissue culture of seed-derived calli carrying Ac and inactive Ds elements. In the F(2) progeny from genetic crosses between the same Ds and Ac starter lines, most of the crosses produced an independent germinal transposition frequency of 10-20%. Also, many Ds elements underwent immobilization even though Ac was expressed. By comparison, in a callus-derived regenerated population, over 70% of plants carried independent Ds insertions, indicating transposition early in callus formation. In the remaining population, the majority of plants carried only Ac. Most of the new Ds insertions were stably transmitted to a subsequent generation. An exceptionally high proportion of independent transposants in the regenerated population means that selection markers for transposed Ds and continual monitoring of Ac/Ds activities may not necessarily be required. By analyzing 1297 Ds-flanking DNA sequences, a genetic map of 1072 Ds insertion sites was developed. The map showed that Ds elements were transposed onto all of the rice chromosomes, with preference not only near donor sites (36%) but also on certain physically unlinked arms. Populations from both genetic crossing and tissue culture showed the same distribution patterns of Ds insertion sites. The information of these mapped Ds insertion sites was deposited in GenBank. Among them, 55% of Ds elements were on predicted open-reading frame (ORF) regions. Thus, we propose an optimal strategy for the rapid generation of a large population of Ds transposants in rice.

Reversible phosphorylation is a key mechanism for the control of intercellular events in eukaryotic cells. In animal cells, Ca2+/CaM-dependent protein phosphorylation and dephosphorylation are implicated in the regulation of a number of cellular processes. However, little is known on the functions of Ca2+/CaM-dependent protein kinases and phosphatases in Ca2+ signaling in plants. From an Arabidopsis expression library, we isolated cDNA encoding a dual specificity protein phosphatase 1, which is capable of hydrolyzing both phosphoserine/threonine and phosphotyrosine residues of the substrates. Using a gel overlay assay, we identified two Ca2+-dependent CaM binding domains (CaMBDI in the N terminus and CaMBDII in the C terminus). Specific binding of CaM to two CaMBD was confirmed by site-directed mutagenesis, a gel mobility shift assay, and a competition assay using a Ca2+/CaM-dependent enzyme. At increasing concentrations of CaM, the biochemical activity of dual specificity protein phosphatase 1 on the p-nitrophenyl phosphate (pNPP) substrate was increased, whereas activity on the phosphotyrosine of myelin basic protein (MBP) was inhibited. Our results collectively indicate that calmodulin differentially regulates the activity of protein phosphatase, dependent on the substrate. Based on these findings, we propose that the Ca2+ signaling pathway is mediated by CaM cross-talks with a protein phosphorylation signal pathway in plants via protein dephosphorylation.

A cDNA encoding RARF1 (rice ADP-ribosylation factor 1) was isolated from fungal elicitor-treated rice suspension culture cells by mRNA differential display. RARF1 transcripts accumulated in response to hydrogen peroxide (H 2 O 2 ) and salicylic acid (SA) and rapidly in cells inoculated with an avirulent pathogen. Constitutively over-expressed RARFI under the control of the cauliflower mosaic virus 35S promoter (CaMV 35S) triggered spontaneous induction of lesion mimics, induced an array of pathogenosis-related (PR) genes, reduced susceptibility to a fungal pathogen, and caused accumulation of SA. From these data, we deduced that RARF1 might be a component of various plant defence signalling pathways involved in inducing the expression of a subset of PR genes.

Transient influx of Ca2+ constitutes an early event in the signaling cascades that trigger plant defense responses. However, the downstream components of defense-associated Ca2+ signaling are largely unknown. Because Ca2+ signals are mediated by Ca2+-binding proteins, including calmodulin (CaM), identification and characterization of CaM-binding proteins elicited by pathogens should provide insights into the mechanism by which Ca2+ regulates defense responses. In this study, we isolated a gene encoding rice Mlo (Oryza sativa Mlo;OsMlo) using a protein-protein interaction-based screening of a cDNA expression library constructed from pathogen-elicited rice suspension cells. OsMlo has a molecular mass of 62 kDa and shares 65% sequence identity and scaffold topology with barley Mlo, a heptahelical transmembrane protein known to function as a negative regulator of broad spectrum disease resistance and leaf cell death. By using gel overlay assays, we showed that OsMlo produced in Escherichia coli binds to soybean CaM isoform-1 (SCaM-1) in a Ca2+-dependent manner. We located a 20-amino acid CaM-binding domain (CaMBD) in the OsMlo C-terminal cytoplasmic tail that is necessary and sufficient for Ca2+-dependent CaM complex formation. Specific binding of the conserved CaMBD to CaM was corroborated by site-directed mutagenesis, a gel mobility shift assay, and a competition assay with a Ca2+/CaM-dependent enzyme. Expression of OsMlo was strongly induced by a fungal pathogen and by plant defense signaling molecules. We propose that binding of Ca2+-loaded CaM to the C-terminal tail may be a common feature of Mlo proteins.

ZAT11, a Zinc Finger of Arabidopsis Thaliana 11, is a dual-function transcriptional regulator that positively regulates primary root growth but negatively regulates Ni (2+) tolerance. Zinc Finger of Arabidopsis Thaliana 11 (ZAT11) is a C2H2-type zinc finger protein that has been reported to function as an active transcriptional repressor. However, the biological function of ZAT11 remains unknown. Here we show that GFP-tagged ZAT11 is targeted to the nucleus. Analysis of plants expressing ZAT11 promoter-GUS showed that ZAT11 is highly expressed in roots and particularly in root tips. To identify the biological function of ZAT11, we constructed three independent lines of ZAT11 overexpressing transgenic plant (ZAT11 OE). ZAT11 OE enhanced the elongation of primary root but reduced the metal tolerance against nickel ion (Ni(2+)). The reduced Ni(2+) tolerance of ZAT11 OE was correlated with decreased accumulation of Ni(2+) in plants. The decreased accumulation of Ni(2+) in ZAT11 OE was caused by the reduced transcription of a vacuolar Ni(2+) transporter gene. Taken together, our results suggest that ZAT11 is a dual function transcriptional regulator that positively regulates primary root growth but negatively regulates Ni(2+) tolerance.

In the interaction between plants and pathogens, carbon (C) resources provide energy and C skeletons to maintain, among many functions, the plant immune system. However, variations in C availability on pathogen associated molecular pattern (PAMP) triggered immunity (PTI) have not been systematically examined. Here, three types of starch mutants with enhanced susceptibility to Pseudomonas syringae pv. tomato DC3000 hrcC were examined for PTI. In a dark period-dependent manner, the mutants showed compromised induction of a PTI marker, and callose accumulation in response to the bacterial PAMP flagellin, flg22. In combination with weakened PTI responses in wild type by inhibition of the TCA cycle, the experiments determined the necessity of C-derived energy in establishing PTI. Global gene expression analyses identified flg22 responsive genes displaying C supply-dependent patterns. Nutrient recycling-related genes were regulated similarly by C-limitation and flg22, indicating re-arrangements of expression programs to redirect resources that establish or strengthen PTI. Ethylene and NAC transcription factors appear to play roles in these processes. Under C-limitation, PTI appears compromised based on suppression of genes required for continued biosynthetic capacity and defenses through flg22. Our results provide a foundation for the intuitive perception of the interplay between plant nutrition status and pathogen defense.

The Ca 2+ signal is essential for the activation of plant defense responses, but downstream components of the signaling pathway are still poorly defined. Here we demonstrate that specific calmodulin (CaM) isoforms are activated by infection or pathogen-derived elicitors and participate in Ca 2+ -mediated induction of plant disease resistance responses. Soybean CaM (SCaM)-4 and SCaM-5 genes, which encode for divergent CaM isoforms, were induced within 30 min by a fungal elicitor or pathogen, whereas other SCaM genes encoding highly conserved CaM isoforms did not show such response. This pathogen-triggered induction of these genes specifically depended on the increase of intracellular Ca 2+ level. Constitutive expression of SCaM-4 and SCaM-5 in transgenic tobacco plants triggered spontaneous induction of lesions and induces an array of systemic acquired resistance (SAR)-associated genes. Surprisingly, these transgenic plants have normal levels of endogenous salicylic acid (SA). Furthermore, coexpression of nahG gene did not block the induction of SAR-associated genes in these transgenic plants, indicating that SA is not involved in the SAR gene induction mediated by SCaM-4 or SCaM-5. The transgenic plants exhibit enhanced resistance to a wide spectrum of virulent and avirulent pathogens, including bacteria, fungi, and virus. These results suggest that specific CaM isoforms are components of a SA-independent signal transduction chain leading to disease resistance.

Phytocystatins are proteinaceous inhibitors of cysteine proteases. They have been implicated in the regulation of plant protein turnover and in defense against pathogens and insects. Here, we have characterized an Arabidopsis phytocystatin family gene, Arabidopsis thaliana phytocystatin 4 (AtCYS4). AtCYS4 was induced by heat stress. The heat shock tolerance of AtCYS4-overexpressing transgenic plants was greater than that of wild-type and cys4 knock-down plants, as measured by fresh weight and root length. Although no heat shock elements were identified in the 5'-flanking region of the AtCYS4 gene, canonical ABA-responsive elements (ABREs) and dehydration-responsive elements (DREs) were found. Transient promoter activity measurements showed that AtCYS4 expression was up-regulated in unstressed protoplasts by co-expression of DRE-binding factor 2s (DREB2s), especially by DREB2C, but not by bZIP transcription factors that bind to ABREs (ABFs, ABI5 and AREBs). DREB2C bound to and activated transcription from the two DREs on the AtCYS4 promoter although some preference was observed for the GCCGAC DRE element over the ACCGAC element. AtCYS4 transcript and protein levels were elevated in transgenic DREB2C overexpression lines with corresponding decline of endogenous cysteine peptidase activity. We propose that AtCYS4 functions in thermotolerance under the control of the DREB2C cascade.

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C(2)H(2)-type zinc finger proteins (ZFPs) play diverse roles in plant response to abiotic stresses. ZAT6, an Arabidopsis C(2)H(2)-type ZFP, has been reported to regulate root development and nutrient stress responses. However, its roles in regulation of abiotic stress response are incompletely known. Here, we demonstrate that salt or osmotic stress triggers a strong increase in ZAT6 expression in leaves. Transgenic plants overexpressing ZAT6 showed improved seed germination under salt and osmotic stress. Intriguingly, ZAT6 interacts with a stress-responsive mitogen-activated protein kinase MPK6 in vitro and in planta. ZAT6 is phosphorylated by both recombinant and plant endogenous MPK6. Serine 8 and serine 223 in ZAT6 were identified as the sites phosphorylated by MPK6. In contrast to wild-type form of ZAT6, overexpression of phosphorylation mutant form did not display significantly enhanced salt and osmotic stress tolerance. Altogether, our results suggest that phosphorylation by MPK6 is required for the functional role of ZAT6 in seed germination under salt and osmotic stress.

The phytohormones abscisic acid (ABA) and gibberellic acid (GA) have antagonistic roles in the control of seed germination and seedling development. We report here that the transcriptional regulator MYB44 has a role in the control of seed germination in Arabidopsis thaliana. High levels of the MYB44 transcript are found in dry seeds but the transcript levels decrease during germination. The decrease in transcript level during germination is inhibited by the GA biosynthesis inhibitor paclobutrazol (PAC). MYB44 is phosphorylated by both recombinant and native forms of MPK3 and MPK6 at Ser(53) and Ser(145). Transgenic overexpression of MYB44 results in increased sensitivity of seed germination to ABA or PAC treatment. The PAC-insensitive germination phenotype of the myb44 mutant is complemented by overexpression of wild type MYB44 but not by overexpression of a mutant protein that lacks the MPK-target serines indicating that phosphorylation of MYB44 by MPKs is required for its biological function.

Mitogen-activated protein kinases (MPKs) are involved in a number of signaling pathways that control plant development and stress tolerance via the phosphorylation of target molecules. However, so far only a limited number of target molecules have been identified. Here, we provide evidence that MYB41 represents a new target of MPK6. MYB41 interacts with MPK6 not only in vitro but also in planta. MYB41 was phosphorylated by recombinant MPK6 as well as by plant MPK6. Ser251 in MYB41 was identified as the site phosphorylated by MPK6. The phosphorylation of MYB41 by MPK6 enhanced its DNA binding to the promoter of a LTP gene. Interestingly, transgenic plants over-expressing MYB41WT showed enhanced salt tolerance, whereas transgenic plants over-expressing MYB41S251A showed decreased salt tolerance during seed germination and initial root growth. These results indicate that the phosphorylation of MYB41 by MPK6 is required for the biological function of MYB41 in salt tolerance.

Our recent work demonstrated that chitin treatment modulated the expression of 118 transcription factor (TF) genes in Arabidopsis. To investigate the potential roles of these TF in chitin signaling and plant defense, we initiated an interaction study among these TF proteins, as well as two chitin-activated mitogen-activated protein kinases (MPK3 and MPK6), using a yeast two-hybrid system. This study revealed interactions among the following proteins: three ethylene-responsive element-binding factors (ERF), five WRKY transcription factors, one scarecrow-like (SCL), and the two MPK, in addition to many other interactions, reflecting a complex TF interaction network. Most of these interactions were subsequently validated by other methods, such as pull-down and in planta bimolecular fluorescence complementation assays. The key node ERF5 was shown to interact with multiple proteins in the network, such as ERF6, ERF8, and SCL13, as well as MPK3 and MPK6. Interestingly, ERF5 appeared to negatively regulate chitin signaling and plant defense against the fungal pathogen Alternaria brassicicola and positively regulate salicylic acid signaling and plant defense against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Therefore, ERF5 may play an important role in plant innate immunity, likely through coordinating chitin and other defense pathways in plants in response to different pathogens.