Change in the chemical form of 137Cs with age in needles of Japanese cedar.

Radiocesium (137Cs) derived from the accident of Fukushima Dai-ichi Nuclear Power Plant remains in forests. Although a large proportion of the 137Cs in forests has been transferred to soils, the rates of transfer to soils depend on the chemical form of 137Cs, which determines the mobility of 137Cs in plant tissues and subsequently during decomposition of leaf litter. In order to understand the dynamics of 137Cs in Sugi (Japanese cedar, Cryptomeria japonica) forests, we identified the chemical forms, such as water soluble, ion-exchangeable, and residual of 137Cs, 133Cs, K, and Rb in needle-bearing Sugi branches of different ages across several years. Compared with the results for K and Rb, Cs (133Cs + 137Cs) tended to change from a water-soluble form to an immobilized form with aging of needle-bearing branch segments. In addition, it was observed that a larger proportion of the immobilized Cs were accumulated in the green outer portions of the stems through aging.

Distributions of cadmium, zinc, and polyphenols in Gamblea innovans.

Gamblea innovans is a Cd- and Zn-accumulating deciduous tree widely distributed in the secondary forests of Japan. We aimed to understand the characteristics of Cd and Zn accumulation in G. innovans in order to effectively utilize the species for phytoremediation. To accomplish that, we studied the relationship between secondary metabolite concentrations and the accumulation and distributions of Cd and Zn in G. innovans leaves and basal stems using micro-X ray fluorescence (µ-XRF). Our results showed a negative correlation between Zn leaf concentrations and polyphenol/2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. This finding might be related to stress or the manifestation of a mechanism for tolerance to Cd and Zn accumulation. In addition, we observed that Cd accumulated primarily in the apoplastic region of surface tissues such as bark and the epidermis of leaves, whereas Zn accumulated in both apoplastic and symplastic regions. Thus, it might be possible that G. innovans can distinguish between Cd and Zn and control their translocation.

Major and minor elemental compositions of streambed biofilms and its implications of riverine biogeochemical cycles.

Chemical compositions of streambed biofilms from a major river of central Japan (the Kushida River) were obtained, with data of associated sediments (fine-grained fractions < 63 µm) and dissolved components of waters, in order to provide preliminary information about biogeochemical significance of streambed biofilms. During the sampling period (July 31st to August 3rd, 2013), dissolved components of the river waters were influenced by the dam reservoir. Concentrations of NO3-, silica (as Si), SO42-, PO43- and Ca2+ decreased across the dam, whereas Fe and Mn increased across the dam, and then decreased downstream rapidly. Streambed biofilms contain significant amount of non-nutrient elements such as Al (up to 21% as Al2O3 on water and others-free basis), indicating that they are contaminated as siliciclatic (silt and clay) materials. Siliciclastic materials in the biofilms are basically compositionally similar to fine-grained (<63 µm) fractions of streambed sediments. However, some elements such as Ca, P, Mn, and Zn are markedly enriched in the biofilms. Particularly, Mn concentrations in the biofilm samples collected just below the dam reservoir are very high (∼4.0 wt %), probably due to accumulation from the discharged water. Concentrations of trace elements such as P, Cr, Cu, Zn and V appear to be controlled by amounts of Fe-oxides and/or Mn-oxides in biofilms. Numbers of factors are involved in controlling chemical compositions of streambed biofilms, including amount of contaminated siliciclastics, authigenic mineral formation, adsorption of dissolved materials and microbial metabolisms. As demonstrated by this study, systematic analyses including major elements and comparison with associated sediments and waters could reveal biogeochemistry of this complex system.

Translocation of 133Cs administered to Cryptomeria japonica wood.

To reveal the in planta behaviour of caesium (Cs), the stable isotope 133Cs was administered into 3-year-old Cryptomeria japonica seedlings by the application of 133CsCl aqueous solution to the bark surface. The administered 133Cs was quantified by ICP-MS measurements, which showed transportation of 133Cs in an ascending direction in the stem. Distribution of 133Cs was visualized using freeze-fixed C. japonica woody stem samples and cryo-time-of-flight secondary ion mass spectrometry/scanning electron microscopy (cryo-TOF-SIMS/SEM) analysis. Cryo-TOF-SIMS/SEM visualization suggested that 133Cs was rapidly transported radially by ray parenchyma cells followed by axial transportation by pith and axial parenchyma cells. Adsorption experiments using powdered C. japonica wood samples and X-ray absorption fine structure (XAFS) analysis suggested that 133Cs was in the hydrated state following its deposition into tracheid cell walls.

Varietal differences in the growth responses of rice to an arbuscular mycorrhizal fungus under natural upland conditions.

Seedlings of three rice (Oryza sativa L.) varieties (one indica, ARC5955; and two japonica, Nipponbare and Koshihikari) with or without pre-colonization by the arbuscular mycorrhizal fungus Funneliformis mosseae were transplanted into an upland field and grown to maturity. Pre-colonization had no effect on the yield of Nipponbare or Koshihikari. However, pre-colonized ARC5955 exhibited a strong tendency toward increased yield, which was accompanied by increases in the percentage of ripened grain and the 1000-grain weight. The rice roots were also colonized by indigenous arbuscular mycorrhizal fungi in the field, but these had only limited effects on shoot biomass and grain yields. We speculate that F. mosseae may have exhibited priority effects, allowing it to dominate the rice roots. There was no significant difference in the contents of most mineral elements in the shoots of pre-colonized ARC5955 at harvest, indicating that some other factor is responsible for the observed yield increase.

Boiogito Increases the Metabolism of Fatty Acids in Proximal Tubular Cells through Peroxisome Proliferators-Activated Receptor (PPAR) α Agonistic Activity.

The promotion of fatty acid metabolism, to which peroxisome proliferators-activated receptor (PPAR) α contributes, has been suggested to participate in maintaining the function of renal proximal tubular epithelial cells (PTECs). The loading of fatty acids to PTECs could result in cell inflammation and cell death. A "Kampo" medicine, Boiogito (BO), is used to treat overweight women exhibiting chronic fatigue and edema in the lower extremities or knees. BO improves renal function by reducing the portion of fatty acids, thereby preventing damage to PTECs. In this study, BO and Astragalus Root (AsR), a constituent crude drug of BO, were administered orally to intravenously bovine serum albumin (BSA)-administered mice to evaluate the PPARα-cAMP responsive element binding protein (CREB) binding protein (CBP) complex binding activity and/or mRNA expression of PPARα, as quantified by enzyme-linked immunosorbent assay (ELISA) and/or polymerase chain reaction (PCR). Increases in PPARα-CBP complex binding activity and the expression of PPARα mRNA were observed not only in BO-administered mice but also in AsR-administered mice, accompanied by a decrease in the amount of renal fatty acid.

Evaluation of microelement contents in Clethra barbinervis as food for human and animals in contrasting geological areas.

The young leaves of Clethra barbinervis Sieb. et Zucc, which is a deciduous tree species found in secondary forests widely in Japan, are used in spring as a local traditional food by local populations, and the bark of this plant is also preferred by sika deer, Cervus nippon. However, C. barbinervis has been known to accumulate heavy metals in its leaves. Then, we aimed to clarify the characteristics of microelement contents in C. barbinervis and to discuss the value of this species as food for humans and animals through the analysis of seasonal changes and distribution in various organs of C. barbinervis growing under two different geological conditions. We found that C. barbinervis is an accumulating and tolerant plant for Ni, Co and Mn. It accumulates Ni from serpentine soil containing Ni at high concentration, and Co and Mn from acidic soils based on crystalline schist. The seasonal variation in element concentrations in leaves indicates that the young leaves contain Cu at high concentration and that eating them in spring season may be advantageous to humans, due to the associated increase in Cu intake. The high concentrations of Cu and Zn in the bark of C. barbinervis might explain why deer prefer to eat the bark of this species.

Can Clethra barbinervis Distinguish Nickel and Cobalt in Uptake and Translocation?

Clethra barbinervis Sieb. et Zucc. accumulates Nickel (Ni) and Cobalt (Co) at high concentrations., We hypothesized that C. barbinervis cannot distinguish between Ni and Co because of the similar chemical properties of these two elements. To confirm this hypothesis and understand the role of these elements in C. barbinervis, we conducted a hydroponic split-root experiment using Ni and Co solutions. We found that the bioconcentration factor (BCF; metal concentration of each tissue/metal concentrations of each treatment solution) of Ni and Co did not significantly differ in the roots, but the BCF for Co was higher than that for Ni in the leaves. The leaves of C. barbinervis accumulated Ni or Co at high concentrations. We also found the simultaneous accumulation of Ni and Co by the multiple heavy metal treatments (Ni and Co) at high concentrations similar to those for the single treatments (Ni or Co). Elevated sulfur concentrations occurred in the roots and leaves of Co-treated seedlings but not in Ni. This result indicates that S was related to Co accumulation in the leaves. These results suggest that C. barbinervis distinguishes between Ni and Co during transport and accumulation in the leaves but not during root uptake.

Characterization of the histidine-rich loop of Arabidopsis vacuolar membrane zinc transporter AtMTP1 as a sensor of zinc level in the cytosol.

The vacuolar Zn(2+)/H(+) antiporter of Arabidopsis thaliana, AtMTP1, has a long cytosolic histidine-rich loop. A mutated AtMTP1 in which the first half of the loop (His-half) was deleted exhibited a 11-fold higher transport velocity in yeast cells. Transgenic lines overexpressing the His-half-deleted AtMTP1 in the loss-of-function mutant were evaluated for growth and metal content in the presence of various zinc concentrations. These overexpressing lines (35S-AtMTP1 and 35S-His-half lines) showed high tolerance to excess concentrations of zinc at 150 µM, as did the wild type, compared with the loss-of-function line. The His-half AtMTP1 transported cobalt in a heterologous expression assay in yeast, but the cumulative amount of cobalt in 35S-His-half plants was not increased. Moreover, the accumulation of calcium and iron was not changed in plants. Under zinc-deficient conditions, growth of 35S-His-half lines was markedly suppressed. Under the same conditions, the 35S-His-half lines accumulated larger amounts of zinc in roots and smaller amounts of zinc in shoots compared with the other lines, suggesting an abnormal accumulation of zinc in the roots of 35S-His-half lines. As a result, the shoots may exhibit zinc deficiency. Taken together, these results suggest that the His-loop acts as a sensor of cytosolic zinc to maintain an essential level in the cytosol and that the dysfunction of the loop results in an uncontrolled accumulation of zinc in the vacuoles of root cells.

Characteristics of a root hair-less line of Arabidopsis thaliana under physiological stresses.

The plasma membrane-associated Ca(2+)-binding protein-2 of Arabidopsis thaliana is involved in the growth of root hair tips. Several transgenic lines that overexpress the 23 residue N-terminal domain of this protein under the control of the root hair-specific EXPANSIN A7 promoter lack root hairs completely. The role of root hairs under normal and stress conditions was examined in one of these root hair-less lines (NR23). Compared with the wild type, NR23 showed a 47% reduction in water absorption, decreased drought tolerance, and a lower ability to adapt to heat. Growth of NR23 was suppressed in media deficient in phosphorus, iron, calcium, zinc, copper, or potassium. Also, the content of an individual mineral in NR23 grown in normal medium, or in medium lacking a specific mineral, was relatively low. In wild-type plants, the primary and lateral roots produce numerous root hairs that become elongated under phosphate-deficient conditions; NR23 did not produce root hairs. Although several isoforms of the plasma membrane phosphate transporters including PHT1;1-PHT1;6 were markedly induced after growth in phosphate-deficient medium, the levels induced in NR23 were less than half those observed in the wild type. In phosphate-deficient medium, the amounts of acid phosphatase, malate, and citrate secreted from NR23 roots were 38, 9, and 16% of the levels secreted from wild-type roots. The present results suggest that root hairs play significant roles in the absorption of water and several minerals, secretion of acid phosphatase(s) and organic acids, and in penetration of the primary roots into gels.

Exploring dynamics of molybdate in living animal cells by a genetically encoded FRET nanosensor.

Molybdenum (Mo) is an essential trace element for almost all living organisms including animals. Mo is used as a catalytic center of molybdo-enzymes for oxidation/reduction reactions of carbon, nitrogen, and sulfur metabolism. Whilst living cells are known to import inorganic molybdate oxyanion from the surrounding environment, the in vivo dynamics of cytosolic molybdate remain poorly understood as no appropriate indicator is available for this trace anion. We here describe a genetically encoded Förester-resonance-energy-transfer (FRET)-based nanosensor composed of CFP, YFP and the bacterial molybdate-sensor protein ModE. The nanosensor MolyProbe containing an optimized peptide-linker responded to nanomolar-range molybdate selectively, and increased YFP:CFP fluorescence intensity ratio by up to 109%. By introduction of the nanosensor, we have been able to successfully demonstrate the real-time dynamics of molybdate in living animal cells. Furthermore, time course analyses of the dynamics suggest that novel oxalate-sensitive- and sulfate-resistant- transporter(s) uptake molybdate in a model culture cell.

Common functions or only phylogenetically related? The large family of PLAC8 motif-containing/PCR genes.

PLAC8 motif-containing proteins form a large family and members can be found in fungi, algae, higher plants and animals. They include the PCR proteins of plants. The name giving PLAC8 domain was originally found in a protein residing in the spongiotrophoblast layer of the placenta of mammals. A further motif found in a large number of these proteins including several PCR proteins is the CCXXXXCPC or CLXXXXCPC motif. Despite their wide distribution our knowledge about the function of these proteins is very limited. For most of them two membrane-spanning α-helices are predicted, indicating that they are membrane associated or membrane intrinsic proteins. In plants PLAC8 motif-containing proteins have been described to be implicated in two very different functions. On one hand, it has been shown that they are involved in the determination of fruit size and cell number. On the other hand, two members of this family, AtPCR1 and AtPCR2 play an important role in transport of heavy metals such as cadmium or zinc. Transport experiments and approaches to model the 3_D structure of these proteins indicate that they could act as transporters for these divalent cations by forming homomultimers. In this minireview we discuss the present knowledge about this protein family and try to give an outlook on how to integrate the different proposed functions into a common picture about the role of PLAC8 motif-containing proteins.

iTRAQ analysis reveals mechanisms of growth defects due to excess zinc in Arabidopsis.

The micronutrient zinc is essential for all living organisms, but it is toxic at high concentrations. Here, to understand the effects of excess zinc on plant cells, we performed an iTRAQ (for isobaric tags for relative and absolute quantification)-based quantitative proteomics approach to analyze microsomal proteins from Arabidopsis (Arabidopsis thaliana) roots. Our approach was sensitive enough to identify 521 proteins, including several membrane proteins. Among them, IRT1, an iron and zinc transporter, and FRO2, a ferric-chelate reductase, increased greatly in response to excess zinc. The expression of these two genes has been previously reported to increase under iron-deficient conditions. Indeed, the concentration of iron was significantly decreased in roots and shoots under excess zinc. Also, seven subunits of the vacuolar H(+)-ATPase (V-ATPase), a proton pump on the tonoplast and endosome, were identified, and three of them decreased significantly in response to excess zinc. In addition, excess zinc in the wild type decreased V-ATPase activity and length of roots and cells to levels comparable to those of the untreated de-etiolated3-1 mutant, which bears a mutation in V-ATPase subunit C. Interestingly, excess zinc led to the formation of branched and abnormally shaped root hairs, a phenotype that correlates with decreased levels of proteins of several root hair-defective mutants. Our results point out mechanisms of growth defects caused by excess zinc in which cross talk between iron and zinc homeostasis and V-ATPase activity might play a central role.

PCaPs, possible regulators of PtdInsP signals on plasma membrane.

In plants, Ca(2+), phosphatidylinositol phosphates (PtdInsPs) and inositol phosphates are major components of intracellular signaling. Several kinds of proteins and enzymes, such as calmodulin (CaM), protein kinase, protein phosphatase, and the Ca(2+) channel, mediate the signaling. Two new Ca(2+)-binding proteins were identified from Arabidopsis thaliana and named PCaP1 and PCaP2 [plasma membrane (PM)-associated Ca(2+) (cation)-binding protein 1 and 2]. PCaP1 has an intrinsically disordered region in the central and C-terminal parts. The PCaP1 gene is expressed in most tissues and the PCaP2 gene is expressed predominantly in root hairs and pollen tubes. We recently demonstrated that these proteins are N-myristoylated, stably anchored in the PM, and are bound with phosphatidylinositol phosphates, especially PtdInsP2s. Here we propose a model for the switching mechanism of Ca (2+)-signaling mediated by PtdInsPs. Ca(2+) forms a complex with CaM (Ca(2+)-CaM) when there is an increase in the cytosol free Ca(2+). The binding of PCaPs with Ca(2+)-CaM causes PCaPs to release PtdInsPs. Until the release of PtdInsPs, the signaling is kept in the resting state.

A mutant strain Arabidopsis thaliana that lacks vacuolar membrane zinc transporter MTP1 revealed the latent tolerance to excessive zinc.

A mutant line of Arabidopsis thaliana that lacks a vacuolar membrane Zn(2+)/H(+) antiporter MTP1 is sensitive to zinc. We examined the physiological changes in this loss-of-function mutant under high-Zn conditions to gain an understanding of the mechanism of adaptation to Zn stress. When grown in excessive Zn and observed using energy-dispersive X-ray analysis, wild-type roots were found to accumulate Zn in vacuolar-like organelles but mutant roots did not. The Zn content of mutant roots, determined by chemical analysis, was one-third that of wild-type roots grown in high-Zn medium. Severe inhibition of root growth was observed in mtp1-1 seedlings in 500 muM ZnSO(4). Suppression of cell division and elongation by excessive Zn was reversible and the cells resumed growth in normal medium. In mutant roots, a marked formation of reactive oxygen species (ROS) appeared in the meristematic zone, where the MTP1 gene was highly expressed. Zn treatment enhanced the expression of several genes involved in Zn tolerance: namely, the plasma membrane Zn(2+)-export ATPase, HMA4, and plasma and vacuolar membrane proton pumps. CuZn-superoxide dismutases, involved in the detoxification of ROS, were also induced. The expression of plasma membrane Zn-uptake transporter, ZIP1, was suppressed. The up- or down-regulation of these genes might confer the resistance to Zn toxicity. These results indicate an essential role of MTP1 in detoxification of excessive Zn and provide novel information on the latent adaptation mechanism to Zn stress, which is hidden by MTP1.

A hydrophilic cation-binding protein of Arabidopsis thaliana, AtPCaP1, is localized to plasma membrane via N-myristoylation and interacts with calmodulin and the phosphatidylinositol phosphates PtdIns(3,4,5)P(3) and PtdIns(3,5)P(2).

A hydrophilic cation-binding protein, PCaP1, was found to be stably bound to the plasma membrane in Arabidopsis thaliana. PCaP1 was quantified to account for 0.03-0.08% of the crude membrane fractions from roots and shoots. Its homologous protein was detected in several plant species. We investigated the mechanism of membrane association of PCaP1 by transient expression of fusion protein with green fluorescent protein. The amino-terminal sequence of 27 residues of PCaP1 had a potential to localize the fusion protein with green fluorescent protein to the plasma membrane, and the substitution of Gly at position 2 with Ala resulted in the cytoplasmic localization of PCaP1. When PCaP1 was expressed in the in vitro transcription/translation system with [(3)H]myristic acid, the label was incorporated into PCaP1, but not into a mutant PCaP1 with Gly2 replaced by Ala. These results indicate that PCaP1 tightly binds to the plasma membrane via N-myristoylation at Gly2. We examined the binding capacity with phosphatidylinositol phosphates (PtdInsPs), and found that PCaP1 selectively interacts with phosphatidylinositol 3,5-bisphosphate and phosphatidylinositol 3,4,5-triphosphate. Competition assay with the N-terminal peptide and mutational analysis revealed that PCaP1 interacts with these two PtdInsPs at the N-terminal part. Interaction of PCaP1 with the membrane and PtdInsPs was not altered in the presence of Ca(2+) at physiological concentrations. Furthermore, calmodulin associated with PCaP1 in a Ca(2+)-dependent manner, and its association weakened the interaction of PCaP1 with PtdInsPs. These results indicate that the N-terminal part is essential for both N-myristoylation and interaction with PtdInsPs, and that PCaP1 may be involved in intracellular signalling through interaction with PtdInsPs and calmodulin.

Enhancement of root respiration and photosynthesis in Quercus serrata Thunb. seedlings by long-term aluminum treatment.

In this article, we discuss the effects of long-term (17 months) aluminum (Al) treatment on the growth, root respiration rate, nutrient uptake rate, and photosynthetic activity of two-year-old Quercus serrata Thunb. seedlings. The seedlings were cultivated by supplying a nutrient solution with or without Al twice a week for 17 months in siliceous sand. After 5- and 17-month treatments, growth, root respiration rate, photosynthetic activity, and the concentration of minerals in the roots and leaves were measured. Al treatment for 5 and 17 months markedly enhanced the growth and photosynthetic activity, and Al treatment for 17 months stimulated the root respiration rate. Although Al treatment for 17 months significantly decreased the Ca, Mg, and P concentrations in leaves, this decrease did not cause deficiency symptoms because of significant increases in shoot biomass and photosynthetic activity. The results of this study reveal that an increase in photosynthetic activity supports growth enhancement induced by Al treatment in Q. serrata seedlings, and suggest that Al may act as a trigger in activating some metabolic functions that can induce growth.

Effect of aluminum on nitrate reductase and photosynthetic activities in Quercus serrata seedlings.

We investigated the physiological effect of rhizospheric aluminum (Al) on the activities of nitrate reductase and photosynthesis in Quercus serrata seedlings. The seedlings were cultured hydroponically in nutrient solution with or without 1 mM AlCl(3) (pH 4.0) for 14 days. After Al treatment for 3 days, the number of primordia of tertiary lateral roots on secondary lateral roots appeared to increase. As a result, the biomass of the roots significantly increased (by 5%) after Al treatment for 14 days. The uptake of NO(3)(-) by the seedlings from the culture medium was stimulated to 125% by Al treatment for 3 days. Al treatments for 7 and 14 days increased the nitrate reductase activities in the roots to 300% and 170%, respectively. Al treatment had no effect on photosynthetic activity or shoot biomass even after 14 days, although the chlorophyll content was slightly increased by Al treatment. These results suggest that the stimulation of root growth by Al might be closely related to metabolic changes including the increase in nitrate reductase activity in the leaves and roots.

Molecular properties of a novel, hydrophilic cation-binding protein associated with the plasma membrane.

A new type of protein was found in Arabidopsis thaliana, PCaP1, which is rich in glutamate and lysine residues. The protein bound (45)Ca(2+) even in the presence of a high concentration of Mg(2+). Real-time polymerase chain reaction and histochemical analysis of promoter-beta-glucuronidase fusions revealed that PCaP1 was expressed in most organs. The PCaP1 protein was detected immunochemically in these organs. Treatment of Arabidopsis seedlings with Cu(2+), sorbitol, or flagellin oligopeptide enhanced the transcription. On the other hand, other sugars, abscisic acid, gibberellic acid, dehydration, and low temperature had little or no effect on PCaP1 transcript abundance. The transient expression of PCaP1 fused to green fluorescent protein in Arabidopsis cells and the subcellular fractionation of tissue homogenate showed that PCaP1 protein is localized to the plasma membrane, although PCaP1 has no predicted transmembrane domain. PCaP1 was associated with the plasma membrane under natural conditions and was released from the membrane at high concentrations of Ca(2+) or Mg(2+) in vitro. These results suggest that the hydrophilic protein PCaP1 binds Ca(2+) and other cations and is stably associated with the plasma membrane.