Identification of tyrosine autophosphorylation sites of Arabidopsis MEKK1 and their involvement in the regulation of kinase activity.

The MEKK1 kinase is a key regulator of stress signaling in Arabidopsis; however, little is known about the regulation of its kinase activity. Here, we found that recombinant MEKK1, expressed in both mammalian HEK293 cells and Escherichia coli, shows a mobility shift in SDS-PAGE, and immunoblotting detected phosphorylation of serine, threonine, and tyrosine residues. N-terminal deletions, site-directed mutagenesis, and protein phosphatase treatment revealed that the mobility shift results from autophosphorylation of the kinase domain. We identified the tyrosine autophosphorylation sites in the N-terminal region of MEKK1. Tyrosine to phenylalanine mutations decrease phosphorylation of the substrate MKK1, suggesting the important role of this residue in the regulation of MEKK1 kinase activity. The present study is the first to show that plant MAPKKKs are regulated by tyrosine phosphorylation.

Control of plant growth and development by overexpressing MAP3K17, an ABA-inducible MAP3K, in Arabidopsis.

Abscisic acid (ABA) plays an important role in plant growth, development, and stress responses. ABA regulates many aspects of plant growth and development, including seed maturation, dormancy, germination, the transition from vegetative to reproductive growth, leaf senescence and responses to environmental stresses, such as drought, high salinity and cold. It is also known that mitogen-activated protein kinase (MAPK) cascades function in ABA signaling. Recently, we and another group have identified the ABA-inducible MAP3Ks MAP3K17 and MAP3K18 as the upstream MAP3Ks of MKK3, implicating the MAP3K17/18-MKK3-MPK1/2/7/14 cascade in ABA signaling. It has also been reported that overexpression of MAP3K18 in Arabidopsis causes an early leaf senescence phenotype, ABA hypersensitive stomata closing, and drought tolerance. In this study, we generated transgenic plants overexpressing MAP3K17 (35S:MAP3K17) and its kinase-inactive form (35S:MAP3K17KN). The bolting of 35S:MAP3K17 was earlier than WT, and the fresh weights of the seedlings were smaller, whereas 35S:MAP3K17KN showed the opposite phenotype. These results indicate that the transition from vegetative to reproductive growth can be regulated by overexpression of MAP3K17 and its kinase-inactive form. Moreover, 35S:MAP3K17 showed lower sensitivity to ABA during post-germinated growth, whereas 35S:MAP3K17 KN showed the opposite phenotype, suggesting the negative roles of MAP3K17 in the response to ABA. Our work provides the possibility to regulate plant growth and development by the genetic manipulation of ABA-induced MAPK cascades, leading to improved crop growth and productivity.

An abscisic acid inducible Arabidopsis MAPKKK, MAPKKK18 regulates leaf senescence via its kinase activity.

Abscisic acid (ABA) is a phytohormone that regulates many physiological functions, such as plant growth, development and stress responses. The MAPK cascade plays an important role in ABA signal transduction. Several MAPK and MAPKK molecules are reported to function in ABA signaling; however, there have been few studies related to the identification of MAPKKK upstream of MAPKK in ABA signaling. In this study, we show that an Arabidopsis MAPKKK, MAPKKK18 functions in ABA signaling. The expression of MAPKKK18 was induced by ABA treatment. Yeast two-hybrid analysis revealed that MAPKKKK18 interacted with MKK3, which interacted with C-group MAPK, MPK1/2/7. Immunoprecipitated kinase assay showed that the 3xFlag-tagged MAPKKK18, expressed in Arabidopsis plants, was activated when treated with ABA. These results indicate the possibility that the MAPK cascade is composed of MAPKKK18, MKK3 and MPK1/2/7 in ABA signaling. The transgenic plants overexpressing MAPKKK18 (35S:MAPKKK18) and its kinase negative mutant (35S:MAPKKK18 KN) were generated, and their growth was monitored. Compared with the WT plant, 35S:MAPKKK18 and 35S:MAPKKK18 KN showed smaller and bigger phenotypes, respectively. Senescence of the rosette leaves was promoted in 35S:MAPKKK18, but suppressed in 35S:MAPKKK18 KN. Furthermore, ABA-induced leaf senescence was accelerated in 35S:MAPKKK18. These results suggest that MAPKKK18 controls the plant growth by adjusting the timing of senescence via its protein kinase activity in ABA dependent manners.

Membrane rigidification functions upstream of the MEKK1-MKK2-MPK4 cascade during cold acclimation in Arabidopsis thaliana.

The MEKK1-MKK2-MPK4 cascade is activated during cold acclimation. However, little is known regarding the perception of low temperature. In this study, we demonstrate that treatment of Arabidopsis with a membrane rigidifier, DMSO, caused MPK4 activation concomitantly with MEKK1 and MKK2 phosphorylation, as well as the cold-inducible gene COR15a expression. These processes are similar to the effects of cold treatment, whereas benzyl alcohol (BA), a membrane fluidizer, prevented such cold-induced events. Moreover, the DMSO-treated seedlings acquired freezing tolerance without cold acclimation. In contrast, the BA-pretreated seedlings did not show freezing tolerance. These results suggest that membrane rigidification activates this MAPK cascade and contributes to the acquisition of freezing tolerance.

Phosphorylation of Arabidopsis thaliana MEKK1 via Ca(2+) signaling as a part of the cold stress response.

The Arabidopsis mitogen activated protein kinase kinase kinase (MEKK1) plays an important role in stress signaling. However, little is known about the upstream pathways of MEKK1. This report describes the regulation of MEKK1 activity during cold signaling. Immunoprecipitated MEKK1 from cold-treated Arabidopsis seedlings showed elevated kinase activity towards mitogen activated protein kinase kinase2 (MKK2), one of the candidate MEKK1 substrates. To clarify how MEKK1 becomes active in response to cold stress signaling, MEKK1 phosphorylation was monitored by an enzyme extracted from the seedlings grown under cold stress with or without EGTA. MEKK1 was phosphorylated after cold stress, but EGTA inhibited the phosphorylation. MKK2 was also phosphorylated by the same extract, but only when EGTA was absent. These results suggested that Ca(2+) signaling occurred upstream of the MEKK1-MKK2 pathway. Full-length MEKK1 showed almost no activity but MEKK1 without the N-terminal region (MEKK1 KD) that retained the kinase domain had a strong ability to phosphorylate MKK2, demonstrating the inhibitory role of the N-terminal region of MEKK1. In addition, MEKK1 was phosphorylated by calcium/calmodulin-regulated receptor-like kinase (CRLK1), which suggested that CRLK1 is one of candidates located upstream of MEKK1.

Global gene profiling and comprehensive bioinformatics analysis of a 46,XY female with pericentric inversion of the Y chromosome.

XY females are rare individuals who carry a Y chromosome but are phenotypically female. In approximately 80-90% of these cases, there are no mutations in the SRY gene, a testis-determining gene on the short arm of the Y chromosome, and the pathophysiology of XY females without SRY mutation remains unclear. In the present study, we used a molecular data mining technique to analyze the pathophysiology of an XY female with functional SRY and pericentric inversion of the Y chromosome, and compared the results with those of a normal male. Interestingly, upregulations of numerous genes included in the development category of the Biological Process ontology, including genes associated with sex determination and organ morphogenesis, were seen in the patient. Additionally, the transforming growth factor-beta (TGF-beta) signaling pathway and Wnt signaling pathway, in which most cell-cell interactions during embryonic development are involved, were altered. Alterations in the expression of numerous genes at the developmental stage, including alterations at both the gene and pathway levels, may persist as a vestige of anomalies of sex differentiation that presumably began in the fetal period. The present study indicates that a data mining technique using bioinformatics contributes to identification of not only genes responsible for birth defects, but also disorders of sex development (DSD)-specific pathways, and that this kind of analysis is an important tool for clarifying the pathophysiology of human idiopathic XY gonadal dysgenesis. Our findings could serve as one of the basic datasets which will be used for future follow-up investigations.

Epigenetic abnormality of SRY gene in the adult XY female with pericentric inversion of the Y chromosome.

In normal ontogenetic development, the expression of the sex-determining region of the Y chromosome (SRY) gene, involved in the first step of male sex differentiation, is spatiotemporally regulated in an elaborate fashion. SRY is expressed in germ cells and Sertoli cells in adult testes. However, only few reports have focused on the expressions of SRY and the other sex-determining genes in both the classical organ developing through these genes (gonad) and the peripheral tissue (skin) of adult XY females. In this study, we examined the gonadal tissue and fibroblasts of a 17-year-old woman suspected of having disorders of sexual differentiation by cytogenetic, histological, and molecular analyses. The patient was found to have the 46,X,inv(Y)(p11.2q11.2) karyotype and streak gonads with abnormally prolonged SRY expression. The sex-determining gene expressions in the patient-derived fibroblasts were significantly changed relative to those from a normal male. Further, the acetylated histone H3 levels in the SRY region were significantly high relative to those of the normal male. As SRY is epistatic in the sex-determination pathway, the prolonged SRY expression possibly induced a destabilizing effect on the expressions of the downstream sex-determining genes. Collectively, alterations in the sex-determining gene expressions persisted in association with disorders of sexual differentiation not only in the streak gonads but also in the skin of the patient. The findings suggest that correct regulation of SRY expression is crucial for normal male sex differentiation, even if SRY is translated normally.

When does the sex ratio of offspring of the paternal 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure decrease: in the spermatozoa stage or at fertilization?

Recent animal experiments confirmed that paternal 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure decreases the sex ratio of offspring at birth without altering litter size. However, the timing of this decrease remained unclear. Male mice were administered TCDD at 7-12 weeks of age and mated with non-treated females. The Y-bearing/X-bearing sperm ratio was examined by real-time PCR and FISH methods, and the sex ratio of the 2-cell embryos collected from non-treated females that had been mated with TCDD-exposed males were investigated by nested PCR. The Y-bearing/X-bearing sperm ratio was not significantly decreased in the TCDD group. However, the sex ratio of the 2-cell embryos of the TCDD group was significantly lower than that of the control group. These results may have resulted from a decrease in fertility of Y-bearing sperm. Thus, the results of this study suggested that the sex ratio of the offspring was decreased at fertilization and not during the spermatozoa stage.

Fetal and neonatal exposure to three typical environmental chemicals with different mechanisms of action: mixed exposure to phenol, phthalate, and dioxin cancels the effects of sole exposure on mouse midbrain dopaminergic nuclei.

A major question is whether exposure to mixtures of low-dose endocrine disruptors (EDs) having different action mechanisms affects neurodevelopment differently than exposure to EDs individually. We therefore investigated the effects of fetal and neonatal exposure to three typical EDs - bisphenol A (BPA), di-(2-ethylhexyl)-phthalate (DEHP), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) - on the midbrain dopaminergic system associated with functions - including motor activity, emotion, and cognition - affected by neuropsychiatric diseases such as attention-deficit/hyperactivity disorder. ICR mouse dams and their pups were orally treated with BPA (5mg/(kg day)), DEHP (1mg/(kg day)), or TCDD (8ng/kg) individually, or with mixtures thereof, to compare the effects between sole and mixed administration. We analyzed tyrosine hydroxylase (TH)- and Fos-immunoreactive (ir) neurons as markers of dopamine and neuronal activation, respectively. The numbers of TH- and/or Fos-ir neurons and the intensity of TH-immunoreactivity within midbrain dopaminergic nuclei (A9, A10, and A8) of each sole administration group significantly differed from controls at 2, 4, and 6 weeks of age. In contrast, no significant differences were detected in the mixture groups, suggesting counteractions among those chemicals. These results indicate that ED mixtures as pollution have unique and elusive effects. Thyroid hormones and/or aryl hydrocarbon receptor-related mechanisms may be responsible for this counteraction.

Posttranscriptional regulation by the upstream open reading frame of the phosphoethanolamine N-methyltransferase gene.

Phosphoethanolamine N-methyltransferase (PEAMT) is involved in choline biosynthesis in plants. The 5' untranslated region (UTR) of several PEAMT genes was found to contain an upstream open reading frame (uORF). We generated transgenic Arabidopsis calli that expressed a chimeric gene constructed by fusing the 5' UTR of the Arabidopsis PEAMT gene (AtNMT1) upstream of the beta-glucuronidase gene. The AtNMT1 uORF was found to be involved in declining levels of the chimeric gene mRNA and repression of downstream beta-glucuronidase gene translation in the calli when the cells were treated with choline. Further, we discuss the role of the uORF.

Activation of Arabidopsis MAPK kinase kinase (AtMEKK1) and induction of AtMEKK1-AtMEK1 pathway by wounding.

We have constructed a series of deletion mutants of Arabidopsis MAPK kinase kinase (AtMEKK1) and obtained a constitutively active mutant, AtMEKK1Delta166, which lacks in self-inhibitory sequence of N-terminal 166 amino acids but still has substrate specificity. AtMEKK1Delta166 predominantly phosphorylates AtMEK1, an Arabidopsis MAPKK, but not its double mutant (AtMEK1T218A/S224E), suggesting that Thr-218 and Ser-224 are the phosphorylation sites. In wounded seedlings, AtMEKK1 was activated and phosphorylated its downstream AtMEK1. Furthermore, analysis using anti-AtMEKK1 and anti-AtMEK1 antibodies revealed that the interaction between the two proteins was signal dependent. These results suggest the presence of AtMEKK1-AtMEK1 pathway induced by wounding.

Similar regulation patterns of choline monooxygenase, phosphoethanolamine N-methyltransferase and S-adenosyl-L-methionine synthetase in leaves of the halophyte Atriplex nummularia L.

Glycinebetaine (betaine) highly accumulates as a compatible solute in certain plants and has been considered to play a role in the protection from salt stress. The betaine biosynthesis pathway of betaine-accumulating plants involves choline monooxygenase (CMO) as the key enzyme and phosphoethanolamine N-methyltransferase (PEAMT), which require S-adenosyl-L-methionine (SAM) as a methyl donor. SAM is synthesized by SAM synthetase (SAMS), and is needed not only for betaine synthesis but also for the synthesis of other compounds, especially lignin. We cloned CMO, PEAMT and SAMS isogenes from a halophyte Atriplex nummularia L. (Chenopodiaceous). The transcript and protein levels of CMO were much higher in leaves and stems than in roots, suggesting that betaine is synthesized mainly in the shoot. The regulation patterns of transcripts for SAMS and PEAMT highly resembled that of CMO in the leaves during and after relief from salt stress, and on a diurnal rhythm. In the leaves, the betaine content was increased but the lignin content was not changed by salt stress. These results suggest that the transcript levels of SAMS are co-regulated with those of PEAMT and CMO to supply SAM for betaine synthesis in the leaves.

A protein kinase activated by darkness phosphorylates nitrate reductase in Komatsuna (Brassica campestris) leaves.

Although it has been shown that leaf nitrate reductase (NR: EC 1.6.6.1) is phosphorylated by subjecting plants to darkness, there is no evidence for the existence of dark-activated or dark-induced NR kinase. This study was undertaken to investigate the occurrence of a protein kinase phosphorylating NR in response to dark treatments. Immediately after transferring Komatsuna (Brassica campestris L.) plants to darkness, we observed rapid increases in the phosphorylating activity of the synthetic peptide, which is designed for the amino acid sequence surrounding the regulatory serine residue of the hinge 1 region of Komatsuna NR, in crude extracts from leaves. The activity reached a maximum after 10 min of darkness. Inactivation states of NR estimated from relative activities with or without Mg2+ were correlated to activities of the putative dark-activated protein kinase. Using the synthetic peptide as a substrate, we purified a protein kinase from dark-treated leaves by means of successive chromatographies on Q-Sepharose, Blue Sepharose, FPLC Q-Sepharose, and ATP-gamma-Sepharose columns. The purified kinase had an apparent molecular mass of 150 kDa with a catalytic subunit of 55 kDa, and it was Ca2+-independent. The purified kinase phosphorylated a recombinant cytochrome c reductase protein, a partial protein of NR, and holo NR, and inactivated NR in the presence of both 14-3-3 protein and Mg2+. The kinase also phosphorylated synthetic peptide substrates designed for sucrose phosphate synthase and 3-hydroxy-3-methylglutaryl-Coenzyme A reductase. Among inhibitors tested, only K252a, a potent and specific serine/threonine kinase inhibitor, completely inhibited the activity of the dark-activated kinase. The activity of the purified kinase was also specifically inhibited by K252a. Taken together with these findings, results obtained suggest that the putative dark-activated protein kinase may be the purified kinase itself, and may be responsible for in vivo phosphorylation of NR and its inactivation during darkness.

Activation of AtMEK1, an Arabidopsis mitogen-activated protein kinase kinase, in vitro and in vivo: analysis of active mutants expressed in E. coli and generation of the active form in stress response in seedlings.

The mitogen-activated protein kinase (MAPK) cascade, consisting of MAPK, MAPK kinase (MAPKK) and MAPK kinase kinase (MAPKKK), is the signaling system that relays various external signals, including mitogens and stresses in eukaryotes. MAPKK is activated by phosphorylation in the consensus motif, SXXXS/T, in animals, but the regulation mechanism for the plant MAPKK by phosphorylation, having the putative phosphorylation motif of S/TXXXXXS/T, is not yet fully clarified. Here we constructed a series of mutants of AtMEK1, an Arabidopsis MAPKK, having the sequence T218-X-S220-X-X-X-S224 that fits both of the plant- and animal-type motifs. We show that the two double-mutant proteins replacing Thr-218/Ser-224 and Ser-220/Ser-224 by Glu expressed in Escherichia coli show a constitutive activity to phosphorylate the Thr and Tyr residues of the kinase-negative mutant of an Arabidopsis MAPK, named ATMPK4, in vitro. The mutation analysis of AtMEK1 replacing Thr-218 and Ser-220 to Ala suggested that Thr-218 is autophosphorylated by the enzyme. The wild-type ATMPK4 was also phosphorylated by the active mutants of AtMEK1 and showed a high protein kinase activity toward myelin basic proteins. In contrast, ATMPK3, another Arabidopsis MAPK, was a poor substrate of this plant MAPKK, indicating that AtMEK1 has a substrate specificity preferring ATMPK4 to ATMPK3, at least in vitro. Furthermore, AtMEK1 immunoprecipitated from Arabidopsis seedlings stimulated with wounding, cold, drought, and high salt showed an elevated protein kinase activity toward the kinase-negative ATMPK4, while the amounts of the AtMEK1 protein did not change significantly. These data indicate that the AtMEK1 becomes an active form through phosphorylation and activates its downstream target ATMPK4 in stress response in Arabidopsis.