Takashi Nanmori

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.

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, AtMEKK1Δ166, which lacks in self-inhibitory sequence of N-terminal 166 amino acids but still has substrate specificity. AtMEKK1Δ166 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.

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 β-glucuronidase gene. The AtNMT1 uORF was found to be involved in declining levels of the chimeric gene mRNA and repression of downstream β-glucuronidase gene translation in the calli when the cells were treated with choline. Further, we discuss the role of the uORF.

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.

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.

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 Ca2+ 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.

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.

Relationship between the Deposition of Phenolic Acids in the Cell Walls and the Cessation of Rapid Growth in Internodes of Floating Rice

Abstract We examined the involvement of p-coumaric, ferulic and 5-5-coupled diferulic acids ester-linked to cell walls in determining the elongation rate of internodes of floating rice (Oryza sativa L.). When floating rice stem segments were exposed to air after 2 days of submergence, the elongation rate of internodes was reduced and the degree of reduction was greater in the light than in the dark, while the internodes of stem segments submerged further for a comparable period continued rapid elongation. The amounts of ferulic and 5-5-coupled diferulic acids in the cell walls in the elongation zone of internodes significantly increased during the first day after exposure to air either in light or darkness. The increase of these phenolics in the cell walls after exposure to air was also observed on the second day in light, but not in darkness. On the other hand, the amount of p-coumaric acid increased only slightly on the first day after exposure to air, but rapidly on the second day in light. This pattern of change in the amounts of p-coumaric acid resembled that in the cell-wall mass (dry weight). The application of sucrose to the segments in darkness increased the amounts of phenolics in the cell walls of internodes to almost the same amount as those in light. These results indicate that the accumulation of ferulic and 5-5-coupled diferulic acids in cell walls may be related to the cessation of internodal elongation in floating rice and that the synthesis of phenolics in the cell wall is caused partially by the provision of sugar in light.

Changes in Cell Wall-Bound Phenolic Acids in the Internodes of Submerged Floating Rice

Abstract Submergence induces rapid elongation of internodes in floating rice (Oryza sativa L.). We examined the distributions of p-coumaric, ferulic and 5-5-coupled diferulic acids ester-linked to cell walls along the axis of highest internodes of submerged and air-grown floating rice stem segments. The amounts of ferulic and 5-5-diferulic acids per cell wall weight were lowest around the intercalary meristem, and increased as the distance from the meristematic zone increased toward the upper part of the internode in both air-grown and submerged stem segments. The ratio of 5-5-diferulic acid to ferulic acid also increased toward the upper, old parts of internodes in both air-grown and submerged stem segments. These observations suggest that the feruloylation of cell wall polysaccharides and the formation of diferulic acids contribute to the cessation of internodal cell elongation and that the formation of diferulic acids in cell walls is controlled by the coupling reaction in addition to the feruloylation. The amounts of p-coumaric acid per unit length and per cell-wall weight were markedly low in the newly elongated region of submerged internodes, and closely correlated with cell-wall dry mass in both air-grown and submerged internodes, suggesting that the deposition of p-coumaric acid in cell walls is related to the formation of secondary cell walls in floating rice internodes.

Identification of the catalytic subunit of cAMP-dependent protein kinase from the photosynthetic flagellate, Euglena gracilis Z.

A gene named epk2 that encodes the amino acid sequence of a protein kinase was identified from the photosynthetic flagellate, Euglena gracilis Z. Homology search and phylogenetic analysis revealed that the deduced amino acid sequence of epk2 is most similar to that of the catalytic subunit of cAMP‐dependent protein kinase (PKA). Northern blot analysis showed that Euglena cells express a 1.4‐kb transcript of this gene. When the EPK2 protein was coexpressed with the rat regulatory subunit of PKA in cultured mammalian cells, these two proteins were coimmunoprecipitated. The association of EPK2 and the rat regulatory subunit of PKA was not detected in the cell lysate incubated with cAMP. EPK2 immunoprecipitated from the transfected cells phosphorylated Kemptide, a synthetic peptide substrate for PKA, and the phosphorylation was inhibited by PKI, a PKA‐selective protein kinase inhibitor. These results indicate that EPK2 is a PKA homologue in the photosynthetic flagellate, and this is the first evidence for the occurrence of the PKA catalytic subunit in photosynthetic organisms.