Rice OsHAK5 is a major potassium transporter that functions in potassium uptake with high specificity but contributes less to cesium uptake.

Cesium (Cs) in the environment is primarily absorbed by a potassium (K) transporter. OsHAK5 is a KT/HAK/KUP family K-transporter showing a high affinity for K. We created cultured rice cells whose OsHAK5 was knocked down by RNAi (named KD). In the medium containing 1.0 m m and less K, the growth of KD was significantly suppressed, suggesting that OsHAK5 greatly contributed to K absorption under limited K conditions. Although Cs suppressed the growth of KD and WT, stronger inhibition was observed on KD. Both KD and WT accumulated similar amounts of Cs when they were cultured in a medium containing Cs, whereas lower amounts of K were detected in KD. These results suggest that OsHAK5 was less involved in the absorption of Cs, although it was essential to K absorption under limited K conditions. In contrast, this means that another transporter may contribute to cesium uptake in rice.

A kinetic analysis of cadmium accumulation in a Cd hyper-accumulator fern, Athyrium yokoscense and tobacco plants.

Cadmium (Cd) accumulations in a Cd hyper-accumulator fern, Athyrium yokoscense (Ay), and tobacco, Nicotiana tabacum (Nt), were kinetically analysed using the positron-emitting tracer imaging system under two medium conditions (basal and no-nutrient). In Ay, maximumly 50% and 15% of the total Cd accumulated in the distal roots and the shoots under the basal condition, respectively. Interestingly, a portion of the Cd in the distal roots returned to the medium. In comparison with Ay, a little fewer Cd accumulations in the distal roots and clearly higher Cd migration to the shoots were observed in Nt under the basal condition (maximumly 40% and 70% of the total Cd, respectively). The no-nutrient condition down-regulated the Cd migration in both species, although the regulation was highly stricter in Ay than in Nt (almost no migration in Ay and around 20% migration in Nt). In addition, the present work enabled to estimate physical and physiological Cd accumulation capacities in the distal roots, and demonstrated condition-dependent changes especially in Ay. These results clearly suggested occurrences of species-/condition-specific regulations in each observed parts. It is probable that integration of these properties govern the specific Cd tolerance/accumulation in Ay and Nt.

Role of Slr1045 in environmental stress tolerance and lipid transport in the cyanobacterium Synechocystis sp. PCC6803.

ATP-binding cassette (ABC) transporter proteins mediate energy-dependent transport of substrates across cell membranes. Numerous ABC transporter-related genes have been found in the Synechocystis sp. PCC6803 genome by genome sequence analysis including H(+), iron, phosphate, polysaccharide, and CO(2) transport-related genes. The substrates of many other ABC transporters are still unknown. To identify ABC transporters involved in acid tolerance, deletion mutants of ABC transporter genes with unknown substrates were screened for acid stress sensitivities in low pH medium. It was found that cells expressing the deletion mutant of slr1045 were more sensitive to acid stress than the wild-type cells. Moreover, slr1045 expression in the wild-type cells was increased under acid stress. These results indicate that slr1045 is an essential gene for survival under acid stress. The mutant displayed high osmotic stress resistance and high/low temperature stress sensitivity. Considering the temperature-sensitive phenotype and homology to the organic solvent-resistant ABC system, we subsequently compared the lipid profiles of slr1045 mutant and wild-type cells by thin-layer chromatography. In acid stress conditions, the phosphatidylglycerol (PG) content in the slr1045 mutant cells was approximately 40% of that in the wild-type cells. Moreover, the addition of PG to the medium compensated for the growth deficiency of the slr1045 mutant cells under acid stress conditions. These data suggest that slr1045 plays a role in the stabilization of cell membranes in challenging environmental conditions. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Slr0967 and Sll0939 induced by the SphR response regulator in Synechocystis sp. PCC 6803 are essential for growth under acid stress conditions.

Two-component signal transduction is the primary signaling mechanism for global regulation of the cellular response to environmental changes. We used DNA microarray analysis to identify genes that were upregulated by acid stress in the cyanobacterium Synechocystis sp. PCC 6803. Several of these genes may be response regulators that are directly involved in this type of stress response. We constructed deletion mutants for the response regulator genes and compared the growth rates of cells transfected with mutant and wild-type genes in a low pH medium. Of these mutants, deletion of sphR affected the growth rate under acid stress (pH 6.0) conditions. We examined genome-wide expression in ΔsphR mutant cells using DNA microarray to determine whether SphR was involved in the regulation of other acid stress responsive genes. Microarray and real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analyses of wild-type cells showed that the expression of phoA, pstS1, and pstS2, which are upregulated under phosphate-limiting conditions, increased (2.48-, 1.88-, and 5.07-fold, respectively) after acid stress treatment for 0.5h. In contrast, pstS2 expression did not increase in the ΔsphR mutant cells after acid stress, whereas the phoA and sphX mRNA levels increased. Furthermore, qRT-PCR and northern blot analysis indicated that downregulation of the acid-responsive genes slr0967 and sll0939 occurred with the deletion of sphR. Indeed, mutants of these genes were more sensitive to acid stress than the wild-type cells. Thus, induction of Slr0967 and Sll0939 by SphR may be essential for growth under acid stress conditions. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Real-time N2O gas detection system for agricultural production using a 4.6-µm-band laser source based on a periodically poled LiNbO3 ridge waveguide.

This article describes a gas monitoring system for detecting nitrous oxide (N2O) gas using a compact mid-infrared laser source based on difference-frequency generation in a quasi-phase-matched LiNbO3 waveguide. We obtained a stable output power of 0.62 mW from a 4.6-µm-band continuous-wave laser source operating at room temperature. This laser source enabled us to detect atmospheric N2O gas at a concentration as low as 35 parts per billion. Using this laser source, we constructed a new real-time in-situ monitoring system for detecting N2O gas emitted from potted plants. A few weeks of monitoring with the developed detection system revealed a strong relationship between nitrogen fertilization and N2O emission. This system is promising for the in-situ long-term monitoring of N2O in agricultural production, and it is also applicable to the detection of other greenhouse gases.

Visualization of the initial radiocesium dynamics after penetration in living apple trees with bark removal using a positron-emitting 127Cs tracer.

Following the Fukushima nuclear accident in March 2011, radiocesium (rCs) contamination in deciduous trees remains over 10 years later even though the trees were leafless at the time of the accident. This phenomenon is considered to be the result of repeated retranslocation of rCs that initially penetrated the bark into the internal tissues. To implement effective measures after a possible accident in the future, it is necessary to clarify how rCs is translocated in the tree after penetration. In this study, rCs translocation was dynamically visualized using a positron-emitting tracer imaging system (PETIS) and autoradiography after the bark of apple branches was removed. The PETIS results showed the translocation of 127Cs from the branch to young shoots and the main stem in apple trees under controlled spring growing conditions. The transport velocity of rCs in the branch was faster than that in the main stem. The transport of rCs, which was either acropetal or basipetal, in the main stem through the branch junction favored basipetal movement. Autoradiography of transverse sections of the main stem indicated that basipetal translocation was due to transport in the phloem. This study demonstrated the initial translocation responses of rCs similar to previous field research, which indicates that rCs transport to the young shoots tends to be higher under controlled conditions. Our laboratory-based experimental system may be useful to gain an improved understanding of rCs dynamics in deciduous trees.

Crystal structure of plant ferritin reveals a novel metal binding site that functions as a transit site for metal transfer in ferritin.

Ferritins are important iron storage and detoxification proteins that are widely distributed in living kingdoms. Because plant ferritin possesses both a ferroxidase site and a ferrihydrite nucleation site, it is a suitable model for studying the mechanism of iron storage in ferritin. This article presents for the first time the crystal structure of a plant ferritin from soybean at 1.8-A resolution. The soybean ferritin 4 (SFER4) had a high structural similarity to vertebrate ferritin, except for the N-terminal extension region, the C-terminal short helix E, and the end of the BC-loop. Similar to the crystal structures of other ferritins, metal binding sites were observed in the iron entry channel, ferroxidase center, and nucleation site of SFER4. In addition to these conventional sites, a novel metal binding site was discovered intermediate between the iron entry channel and the ferroxidase site. This site was coordinated by the acidic side chain of Glu(173) and carbonyl oxygen of Thr(168), which correspond, respectively, to Glu(140) and Thr(135) of human H chain ferritin according to their sequences. A comparison of the ferroxidase activities of the native and the E173A mutant of SFER4 clearly showed a delay in the iron oxidation rate of the mutant. This indicated that the glutamate residue functions as a transit site of iron from the 3-fold entry channel to the ferroxidase site, which may be universal among ferritins.

Role of H-1 and H-2 subunits of soybean seed ferritin in oxidative deposition of iron in protein.

Naturally occurring phytoferritin is a heteropolymer consisting of two different H-type subunits, H-1 and H-2. Prior to this study, however, the function of the two subunits in oxidative deposition of iron in ferritin was unknown. The data show that, upon aerobic addition of 48-200 Fe(2+)/shell to apoferritin, iron oxidation occurs only at the diiron ferroxidase center of recombinant H1 (rH-1). In addition to the diiron ferroxidase mechanism, such oxidation is catalyzed by the extension peptide (a specific domain found in phytoferritin) of rH-2, because the H-1 subunit is able to remove Fe(3+) from the center to the inner cavity better than the H-2 subunit. These findings support the idea that the H-1 and H-2 subunits play different roles in iron mineralization in protein. Interestingly, at medium iron loading (200 irons/shell), wild-type (WT) soybean seed ferritin (SSF) exhibits a stronger activity in catalyzing iron oxidation (1.10 ± 0.13 µm iron/subunit/s) than rH-1 (0.59 ± 0.07 µm iron/subunit/s) and rH-2 (0.48 ± 0.04 µm iron/subunit/s), demonstrating that a synergistic interaction exists between the H-1 and H-2 subunits in SSF during iron mineralization. Such synergistic interaction becomes considerably stronger at high iron loading (400 irons/shell) as indicated by the observation that the iron oxidation activity of WT SSF is ∼10 times larger than those of rH-1 and rH-2. This helps elucidate the widespread occurrence of heteropolymeric ferritins in plants.

A novel EP-involved pathway for iron release from soya bean seed ferritin.

Iron in phytoferritin from legume seeds is required for seedling germination and early growth. However, the mechanism by which phytoferritin regulates its iron complement to these physiological processes remains unknown. In the present study, protein degradation is found to occur in purified SSF (soya bean seed ferritin) (consisting of H-1 and H-2 subunits) during storage, consistent with previous results that such degradation also occurs during seedling germination. In contrast, no degradation is observed with animal ferritin under identical conditions, suggesting that SSF autodegradation might be due to the EP (extension peptide) on the exterior surface of the protein, a specific domain found only in phytoferritin. Indeed, EP-deleted SSF becomes stable, confirming the above hypothesis. Further support comes from a protease activity assay showing that EP-1 (corresponding to the EP of the H-1 subunit) exhibits significant serine protease-like activity, whereas the activity of EP-2 (corresponding to the EP of the H-2 subunit) is much weaker. Consistent with the observation above, rH-1 (recombinant H-1 ferritin) is prone to degradation, whereas its analogue, rH-2, becomes very stable under identical conditions. This demonstrates that SSF degradation mainly originates from the serine protease-like activity of EP-1. Associated with EP degradation is a considerable increase in the rate of iron release from SSF induced by ascorbate in the amyloplast (pH range, 5.8-6.1). Thus phytoferritin may have facilitated the evolution of the specific domain to control its iron complement in response to cell iron need in the seedling stage.

Condylar shape in relation to anterior disk displacement in juvenile females.

The purpose of the study was to test the hypothesis that condylar shape varies based upon the condition of anterior disk displacement in young adolescent patients with temporomandibular disorder (TMD). The study design consisted of 96 juvenile female patients (aged 9 to 20; 15.1 +/- 2.3 yrs.) with clinical signs and/or symptoms of TMD. Bilateral high-resolution magnetic resonance imaging scans were performed in frontal and horizontal views with the mandible in the closed position. Disk positions were evaluated to classify the patients into three diagnostic groups. The results of the study, using ANOVA and Bonferroni tests, demonstrated significant differences among the groups. The conclusion drawn from the study was that condylar shape and size vary based on anterior disk position in juvenile females with TMD. The study's results suggest that disk displacement results in a smaller condyle.

The universal mechanism for iron translocation to the ferroxidase site in ferritin, which is mediated by the well conserved transit site.

Ferritins are ubiquitous iron storage proteins. Recently, we identified a novel metal-binding site, transit site, in the crystal structure of phytoferritin. To elucidate the function of the transit site in ferritin from other species, we prepared transit-site-deficient mutants of human H ferritin, E140A and E140Q, and their iron oxidation kinetics was analyzed. The initial velocities of iron oxidization were reduced in the variants, especially in E140Q. The crystal structure of E140Q showed that the side chain of the mutated Gln140 was fixed by a hydrogen bond, whereas that of native Glu140 was flexible. These results suggest that the conserved transit site also has a function to assist with the metal ion sequestration to the ferroxidase site in ferritins from vertebrates.

Nicotinate/nicotinamide mononucleotide adenyltransferase-mediated regulation of NAD biosynthesis protects guard cells from reactive oxygen species in ABA-mediated stomatal movement in Arabidopsis.

Nicotinamide adenine dinucleotide (NAD) and its derivative nicotinamide adenine dinucleotide phosphate (NADP) are indispensable co-factors in broad-spectrum metabolic events for the maintenance of cellular homeostasis in all living organisms. In this study, the cellular expression levels of NAD biosynthesis genes in Arabidopsis were investigated. A very high expression of nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT) was observed in the differentiated stomatal guard cells of the leaf surface. Transcriptional analysis confirmed that several genes in the biosynthesis pathway were also highly expressed in stomatal guard cells. In fact, NAD and NADP metabolisms were investigated during stomatal movement. Importantly, the generation of phytohormone ABA-induced reactive oxygen species, which acts as a signal for stomatal closure, was accompanied by markedly decreased levels of NAD. The ABA-induced oxidative stress caused stomatal cell death in the nmnat mutant. Furthermore, stomata partially lost their ability to close leading to drought susceptibility. The stomata were less responsive to opening cues as well. These results indicate that NAD biosynthesis is involved in protecting guard cells from ABA-induced local oxidative stress via the regulation of NMNAT activity. In this study, it is demonstrated that NMNAT is essential for the maintenance of NAD homeostasis enabling sustainable stomatal movement.

Chemical Speciation, Plant Uptake, and Toxicity of Heavy Metals in Agricultural Soils.

Heavy metals in agricultural soils exist in diverse dissolved (free cations and complexed species of positive, neutral, or negative charges), particulate (sorbed, structural, and coprecipitated), and colloidal (micro- and nanometer-sized particles) species. The fate of different heavy metal species is controlled by the master variables: pH (solubility), ionic strength (activity and charge-shielding), and dissolved organic carbon (complexation). In the rhizosphere, chemical speciation controls toxicokinetics (uptake and transport of metals by plants) while toxicodynamics (interaction between the plant and absorbed species) drives the toxicity outcome. Based on the critical review, the authors recommend omics and data mining techniques to link discrete knowledge bases from the speciation dynamics, soil microbiome, and plant transporter/gene expression relevant to homeostasis conditions of modern agriculture. Such efforts could offer a disruptive application tool to improve and sustain plant tolerance, food safety, and environmental quality.

De novo transcriptome analysis reveals an unperturbed transcriptome under high cadmium conditions in the Cd-hypertolerant fern Athyrium yokoscense.

Athyrium yokoscense shows strong tolerance to cadmium exposure, even at levels that are many times greater than the toxic levels in ordinary plants. To determine the mechanism of Cd tolerance in A. yokoscense, we grew these plants under high Cd conditions and observed the tissue-specific accumulation of Cd and generation of reactive oxygen species, which is one of the major physiological responses to Cd stress. Fuchsin staining indicated the existence of a casparian strip in A. yokoscense roots, which may participate in Cd hypertolerance in A. yokoscense. Moreover, we performed RNA-seq of RNA samples from A. yokoscense plants treated with or without Cd exposure and obtained comprehensive RNA sequences as well as the Cd-responsive expression patterns of individual genes. Through de novo transcriptome assembly and gene expression analysis, we found that A. yokoscense showed normal features with no significant change in the expression levels of any transporter genes, even under high Cd exposure conditions. Our results demonstrate that A. yokoscense has an unusual mechanism that allows the invading Cd to partition into the distal roots, thus avoiding translocation of Cd into the xylem.

Occlusal disharmony increases stress response in rats.

Repeated or chronic stress is known to produce structural and functional changes in the rat brain, and in particular, alter the response of the hypothalamic -- pituitary -- adrenal (HPA) axis to subsequent new stress. Occlusal disharmony via placement of acryl cap on the lower incisors of rats is perceived as chronic stress. To determine the response of the HPA axis to subsequent new stress in rats with occlusal disharmony, we measured plasma corticosterone levels in these rats after subjecting them to new stress. Plasma corticosterone levels in rats with and without incisal cap increased and reached a peak 30 min after exposure to the new stress. However, a later decrease in plasma corticosterone levels from peak levels was found in rats with incisal cap compared with rats without incisal cap. This finding suggests that occlusal disharmony alters the response of the HPA axis to subsequent new stress.

Daily life habits associated with eveningness lead to a higher prevalence of dental caries in children.

BACKGROUND/PURPOSE: Circadian rhythm is an endogenous daily variation observed in most physiological functions including salivary secretion. Irregular lifestyle causes many diseases such as obesity and sleep disorders. The aim of this study is to examine the effects of the timings of sleep and meal on the prevalence of dental caries. MATERIALS AND METHODS: Study was conducted at university hospital in Japan. We asked 230 children (1-16 years old) to record the following life habits for 8 days: waking time, bedtime, mealtimes, snacking frequency, and tooth brushing frequency. We analyzed sleep habits from all data and compared dental caries and life habits using data from subjects with primary (2-7 years old) or permanent (11-16 years old) dentition period. RESULTS: The number of dental caries assessed using the decay or filled teeth (dft) index correlated with bedtime, supper time, regularity of supper time, and snacking frequency in subjects with primary dentition. Multiple regression analysis revealed that bedtime and snacking frequency were mutually independent risk factors for dental caries. No correlations were found between the prevalence of dental caries and other measurement items. The number of caries correlated with the regularity of supper time and age in subjects with permanent dentition. CONCLUSION: Children with daily life habits associated with eveningness have a higher prevalence of dental caries.

Copper treatment of peach leaves causes lesion formation similar to the biotic stress response.

Peach (Prunus persica (L.) Batsch) leaves are sensitive to copper (Cu) exposure. The symptoms of Cu exposure are similar to those of bacterial spot disease; however, the mechanism underlying lesion formation caused by Cu exposure is not clear. Here, we investigated whether lesion formation caused by Cu exposure was related to the mechanism underlying plant resistance to microbial pathogens. When Cu was applied to the centre of a pinhole on peach leaves, a two-step process was observed. A pale green section in the shape of a doughnut, located far from a Cu treatment point, first appeared on a leaf treated with 2 mM CuSO4. Next, a yellow-white section gradually spread from the Cu treatment point to the pale green section. Finally, a gap was formed in the middle of the pale green section. The inner part of the pale green section contained 96% of the Cu applied, indicating that Cu is retained in the lesion area. Real-time PCR analysis of the expression of genes encoding pathogenesis-related proteins and enzymes involved in phytoalexin synthesis revealed that three genes (encoding chitinase, pathogenesis-related protein 4, and ß-1,3-glucanase-3) of the eight tested were upregulated by Cu treatment. Furthermore, treatment with caspase-1 inhibitors reduced lesion formation. These results show that Cu treatment of peach leaves causes cell death similar to that occurring during the biotic stress response.

A through year behavior of 137Cs in a Japanese flowering cherry tree in relation to that of potassium.

To understand the transfer of radiocesium (137Cs) in inside of deciduous trees, changes in 137Cs activity concentrations, primarily derived from the Fukushima accident in March 2011, were observed in the upper parts of a Japanese flowering cherry tree (Prunus x yedoensis cv. Somei-Yoshino) between 2015 and 2018. The sampling of the foliar parts occurred over the entire leaf life span from winter bud to litterfall and those of the branches were distinguished based on emergence years (2017, 2016, 2015, 2014-2011, and 2010/before). First, every tissue demonstrated a clear seasonal variation in 137Cs activity concentration. Second, a synchrony of seasonal variations in 137Cs activity concentration with those in the biological analogue of K concentration was observed in foliar parts during their growth season, but not in branches nor during the other seasons. With respect to the timing of changes in each tissue with tree phenology, it is possible that K and 137Cs alternate between leaves and branches via the same translocation mechanisms. The resorption efficiencies (i.e., 1 - [the concentrations in the last litterfall]/[the maximum concentrations in green leaves]) of K and 137Cs were 76% and 46% in average, respectively. In addition, both leaf buds and branches played an important role as reservoirs during dormancy. The buds storage ratio before and after bud burst (i.e., [the inventories in buds at the end of defoliation]/[those before and after bud burst]) for K were 0.57 and 0.10 in median, respectively, and those for and 137Cs were 1.14 and 0.14 in median, respectively. Consequently, the transfer of 137Cs in inside of trees was still visible seven years after deposition, even though the annual reduction in 137Cs activity concentration was apparent in each tissue.

Assessment of gamma radiation from a limited area of forest floor using a cumulative personal dosimeter.

To elucidate long term changes in gamma radiation from a limited region of interest of the forest floor, a simple monitoring procedure using a cumulative personal dosimeter (D-shuttle) was examined from 2016 to 2017. The test site was in a small forest in Abiko, Japan, where the initial radiocesium contamination from the Fukushima Dai-ichi Nuclear Power Plant was 60-100 kBq m-2. Three experimental plots basically containing a set of two 5 × 5 m2 observation areas were arranged at the site. The litterfall and decomposing organic layer of one area (D: decontaminated) were fully eliminated before the monitoring, whereas the other area (N: natural) was left unchanged. Five D-shuttle sets (i.e., D-shuttle, lead shield, and holder) per area were set up. One D-shuttle set could monitor the specific gamma radiation from radiocesium distributed within a limited area of ground (0.5 m radius of circle = ca. 0.8 m2 area of flat ground). The results indicated significant differences in the accumulated doses among each of the plots and areas, reflecting their soil radiocesium inventories. Interestingly, every index decreased with time, but the decreases were slower than the theoretical decay of radiocesium (134Cs and 137Cs). In addition, the accumulated dose decreased during heavy rainfall events. One possible explanation for these changes of the accumulated dose is a combination of meteorological and tree phenological phenomena, such as radiocesium from the forest canopy being newly added to the floor primarily by litterfall and soil moisture content disturbing radiation emitted from soils. This simple procedure enables long-term observation of gamma radiation from a limited area of forest floor non-invasively and semi-quantitatively.

Repeated immobilization stress in the early postnatal period increases stress response in adult rats.

Repeated immobilization stress tests in the early postnatal period were performed to determine the effects on the growth of developing rats as well as the response of the HPA axis to subsequent novel stress in adulthood. In addition, effects of maternal deprivation (MD) with the same period of the exposure to immobilization stress were also examined. We used 2 different types of immobilization stress and 2 different types of MD: immobilization stress for 30 min/day from postnatal day 7 (P7) to P13 (IS7-13 group); immobilization stress for 30 min on P7 (IS7 group); MD for 30 min/day from P7 to P13 (MD7-13 group); and MD for 30 min on P7 (MD7 group). Body weights were lower in the IS7-13 group than in the control group from P10 to P50, although body weight gain in the MD7-13 group was only transiently affected. Stress-induced corticosterone levels in the IS7-13 group were higher than in the control group and did not return to baseline levels until at least 120 min after the termination of stress, whereas temporal variations of stress-induced corticosterone levels did not differ between the IS, MD7-13, MD7, and control groups. Repeated immobilization stress in the early postnatal period induced long-term effects on the growth of developing rats and stress response of the HPA axis to the novel stress in adulthood, although a single immobilization stress, periodic MD, or a single MD had little effect.