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In addition to the well-known motor control, the cerebellum has recently been implicated in memory, cognition, addiction, and social behavior. Given that the cerebellum contains more neurons than the cerebral cortex and has tight connections to the thalamus and brainstem nuclei, it is possible that the cerebellum also regulates sleep/wakefulness. However, the role of the cerebellum in sleep was unclear, since cerebellar lesion studies inevitably involved massive inflammation in the adjacent brainstem, and sleep changes in lesion studies were not consistent with each other. Here, we examine the role of the cerebellum in sleep and wakefulness using mesencephalon- and rhombomere 1-specific Ptf1a conditional knockout (Ptf1a cKO) mice, which lack the cerebellar cortex and its related structures, and exhibit ataxic gait. Ptf1a cKO mice had similar wake and non-rapid eye movement sleep (NREMS) time as control mice and showed reduced slow wave activity during wakefulness, NREMS and REMS. Ptf1a cKO mice showed a decrease in REMS time during the light phase and had increased NREMS delta power in response to 6 h of sleep deprivation, as did control mice. Ptf1a cKO mice also had similar numbers of sleep spindles and fear memories as control mice. Thus, the cerebellum does not appear to play a major role in sleep-wake control, but may be involved in the generation of slow waves.

Phosphorus (P) is an essential macronutrient for plant growth. In deciduous trees, P is remobilized from senescing leaves and stored in perennial tissues during winter for further growth. Annual internal recycling and accumulation of P are considered an important strategy to support the vigorous growth of trees. However, the pathways of seasonal re-translocation of P and the molecular mechanisms of this transport have not been clarified. Here we show the seasonal P re-translocation route visualized using real-time radioisotope imaging and the macro- and micro-autoradiography. We analysed the seasonal re-translocation P in poplar (Populus alba. L) cultivated under 'a shortened annual cycle system', which mimicked seasonal phenology in a laboratory. From growing to senescing season, sink tissues of

Understanding molecular mechanisms which underlie transport of cesium (Cs+) in plants is important to limit entry of its radioisotopes from contaminated area to the food chain. The potentially toxic element Cs+, which is not involved in any biological process, is chemically closed to the macronutrient potassium (K+). Among the multiple K+ carriers, the high-affinity K+ transporters family HAK/KT/KUP is thought to be relevant in mediating opportunistic Cs+ transport. On the 13 KUP identified in Arabidopsis thaliana, only HAK5, the major contributor to root K+ acquisition under low K+ supply, has been functionally demonstrated to be involved in Cs+ uptake in planta. In the present study, we showed that accumulation of Cs+ increased by up to 30% in two A. thaliana mutant lines lacking KUP9 and grown under low K+ supply. Since further experiments revealed that Cs+ release from contaminated plants to the external medium is proportionally lower in the two kup9 mutants, we proposed that KUP9 disruption could impair Cs+ efflux. By contrast, we did not measure significant impairment of K+ status in kup9 mutants suggesting that KUP9 disruption does not alter substantially K+ transport in experimental conditions used here. Putative primary role of KUP9 in plants is further discussed.

Progress has been made in the elucidation of sleep and wakefulness regulation at the neurocircuit level

Dysbiosis of the gut microbiota affects physiological processes, including brain functions, by altering the intestinal metabolism. Here we examined the effects of the gut microbiota on sleep/wake regulation. C57BL/6 male mice were treated with broad-spectrum antibiotics for 4 weeks to deplete their gut microbiota. Metabolome profiling of cecal contents in antibiotic-induced microbiota-depleted (AIMD) and control mice showed significant variations in the metabolism of amino acids and vitamins related to neurotransmission, including depletion of serotonin and vitamin B6, in the AIMD mice. Sleep analysis based on electroencephalogram and electromyogram recordings revealed that AIMD mice spent significantly less time in non-rapid eye movement sleep (NREMS) during the light phase while spending more time in NREMS and rapid eye movement sleep (REMS) during the dark phase. The number of REMS episodes seen in AIMD mice increased during both light and dark phases, and this was accompanied by frequent transitions from NREMS to REMS. In addition, the theta power density during REMS was lower in AIMD mice during the light phase compared with that in the controls. Consequently, the gut microbiota is suggested to affect the sleep/wake architecture by altering the intestinal balance of neurotransmitters.

Phosphorus (P) is an essential macronutrient for plant growth. In deciduous trees, P is remobilized from senescing leaves and stored in perennial tissues during winter for further growth. Annual internal recycling and accumulation of P is considered an important strategy to support vigorous growth of trees. However, the pathways of seasonal re-translocation of P and the molecular mechanisms of this transport have not been clarified. Here we show the seasonal P re-translocation route visualized using the real-time radioisotope imaging and the macro- and micro-autoradiography. We analyzed the seasonal re-translocation P in poplar (Populus alba. L) cultivated under “a shortened annual cycle system”, which mimicked seasonal phenology in a laboratory. From growing to senescing season, sink tissues of 32P and/or 33P shifted from young leaves and the apex to the lower stem and roots. The radioisotope P re-translocated from a leaf was stored in phloem and xylem parenchyma cells and redistributed to new shoots after dormancy. Seasonal expression profile of phosphate transporters (PHT1, PHT5 and PHO1 family) was obtained in the same system. Our results reveal the seasonal P re-translocation routes at the organ and tissue levels and provide a foothold for elucidating its molecular mechanisms.

Recent studies have shown various metabolic and transcriptomic interactions between sulfur (S) and phosphorus (P) in plants. However, most studies have focused on the effects of phosphate (Pi) availability and P signaling pathways on S homeostasis, whereas the effects of S availability on P homeostasis remain largely unknown. In this study, we investigated the interactions between S and P from the perspective of S availability. We investigated the effects of S availability on Pi uptake, transport, and accumulation in Arabidopsis thaliana grown under sulfur sufficiency (+S) and deficiency (−S). Total P in shoots was significantly increased under −S owing to higher Pi accumulation. This accumulation was facilitated by increased Pi uptake under −S. In addition, −S increased root-to-shoot Pi transport, which was indicated by the increased Pi levels in xylem sap under −S. The −S-increased Pi level in the xylem sap was diminished in the disruption lines of PHT1;9 and PHO1, which are involved in root-to-shoot Pi transport. Our findings indicate a new aspect of the interaction between S and P by listing the increased Pi accumulation as part of −S responses and by highlighting the effects of −S on Pi uptake, transport, and homeostasis.

Phosphorus (P) is a key macronutrient whose availability has a profound effect on plant growth and productivity. The understanding of the mechanism underlying P availability-responsive P acquisition has expanded largely in the past decade; however, effects of other environmental factors on P acquisition and utilization remain elusive. Here, by imaging natural variation in phosphate uptake in 200 natural accessions of Arabidopsis, we identify two accessions with low phosphate uptake activity, Lm-2 and CSHL-5. In these accessions, natural variants of phytochrome B were found to cause both decreased light sensitivity and lower phosphate uptake. Furthermore, we also found that expression levels of phosphate starvation-responsive genes are directly modulated by phytochrome interacting factors (PIF) PIF4/PIF5 and HY5 transcription factors whose activity is under the control of phytochromes. These findings disclose a new molecular mechanism underlying red-light-induced activation of phosphate uptake, which is responsible for different activity for P acquisition in some natural accessions of Arabidopsis. Plants develop shoots and roots to access light, carbon dioxide, water and nutrients. Light intensity and quality are suggested to affect root nutrient uptake. Now, the researchers identify a mechanistic link between red light and phosphorus uptake by investigating 200 natural accessions of Arabidopsis.

Summary: The mammalian brain undergoes sexual differentiation by gonadal hormones during the perinatal critical period. However, the machinery at earlier stages has not been well studied. We found that Ptf1a is expressed in certain neuroepithelial cells and immature neurons around the third ventricle that give rise to various neurons in several hypothalamic nuclei. We show that conditional Ptf1a-deficient mice (Ptf1a cKO) exhibit abnormalities in sex-biased behaviors and reproductive organs in both sexes. Gonadal hormone administration to gonadectomized animals revealed that the abnormal behavior is caused by disorganized sexual development of the knockout brain. Accordingly, expression of sex-biased genes was severely altered in the cKO hypothalamus. In particular, Kiss1, important for sexual differentiation of the brain, was drastically reduced in the cKO hypothalamus, which may contribute to the observed phenotypes in the Ptf1a cKO. These findings suggest that forebrain Ptf1a is one of the earliest regulators for sexual differentiation of the brain. : Fujiyama et al. find that forebrain-specific Ptf1a-deficient mice (Ptf1a cKO) exhibit abnormalities in sexually dimorphic behaviors, reproductive organs, and severely altered expression of sex-biased genes, including Kiss1, in the hypothalamus in both sexes, which suggests that forebrain Ptf1a is one of the earliest regulators for sexual differentiation of the brain. Keywords: sexual differentiation, sexual behavior, hypothalamus, Ptf1a, kisspeptin, central nervous system

Sleep and wake have global effects on brain physiology, from molecular changes1–4 and neuronal activities to synaptic plasticity3–7. Sleep–wake homeostasis is maintained by the generation of a sleep need that accumulates during waking and dissipates during sleep8–11. Here we investigate the molecular basis of sleep need using quantitative phosphoproteomic analysis of the sleep-deprived and Sleepy mouse models of increased sleep need. Sleep deprivation induces cumulative phosphorylation of the brain proteome, which dissipates during sleep. Sleepy mice, owing to a gain-of-function mutation in the Sik3 gene 12 , have a constitutively high sleep need despite increased sleep amount. The brain proteome of these mice exhibits hyperphosphorylation, similar to that seen in the brain of sleep-deprived mice. Comparison of the two models identifies 80 mostly synaptic sleep-need-index phosphoproteins (SNIPPs), in which phosphorylation states closely parallel changes of sleep need. SLEEPY, the mutant SIK3 protein, preferentially associates with and phosphorylates SNIPPs. Inhibition of SIK3 activity reduces phosphorylation of SNIPPs and slow wave activity during non-rapid-eye-movement sleep, the best known measurable index of sleep need, in both Sleepy mice and sleep-deprived wild-type mice. Our results suggest that phosphorylation of SNIPPs accumulates and dissipates in relation to sleep need, and therefore SNIPP phosphorylation is a molecular signature of sleep need. Whereas waking encodes memories by potentiating synapses, sleep consolidates memories and restores synaptic homeostasis by globally downscaling excitatory synapses4–6. Thus, the phosphorylation–dephosphorylation cycle of SNIPPs may represent a major regulatory mechanism that underlies both synaptic homeostasis and sleep–wake homeostasis.

The regulatory network of genes and molecules in sleep/wakefulness remains to be elucidated. Here we describe the methodology and workflow of the dominant screening of randomly mutagenized mice and discuss theoretical basis of forward genetics research for sleep in mice. Our high-throughput screening employs electroencephalogram (EEG) and electromyogram (EMG) to stage vigilance states into a wake, rapid eye movement sleep (REMS) and non-REM sleep (NREMS). Based on their near-identical sleep/wake behavior, C57BL/6J (B6J) and C57BL/6N (B6N) are chosen as mutagenized and counter strains, respectively. The total time spent in the wake and NREMS, as well as the REMS episode duration, shows sufficient reproducibility with small coefficients of variance, indicating that these parameters are most suitable for quantitative phenotype-driven screening. Coarse linkage analysis of the quantitative trait, combined with whole-exome sequencing, can identify the gene mutation associated with sleep abnormality. Our simulations calculate the achievable LOD score as a function of the phenotype strength and the numbers of mice examined. A pedigree showing a mild decrease in total wake time resulting from a heterozygous point mutation in the Cacna1a gene is described as an example.

Recent studies have shown various metabolic and transcriptomic interactions between sulfur (S) and phosphorus (P) in plants. However, most studies have focused on the effects of phosphate (Pi) availability and P signaling pathways on S homeostasis, whereas the effects of S availability on P homeostasis remain largely unknown. In this study, we investigated the interactions between S and P from the perspective of S availability. We investigated the effects of S availability on Pi uptake, transport, and accumulation in Arabidopsis thaliana grown under sulfur sufficiency (+S) and deficiency (&minus, S). Total P in shoots was significantly increased under &minus, S owing to higher Pi accumulation. This accumulation was facilitated by increased Pi uptake under &minus, S. In addition, &minus, S increased root-to-shoot Pi transport, which was indicated by the increased Pi levels in xylem sap under &minus, S. The &minus, S-increased Pi level in the xylem sap was diminished in the disruption lines of PHT1, 9 and PHO1, which are involved in root-to-shoot Pi transport. Our findings indicate a new aspect of the interaction between S and P by listing the increased Pi accumulation as part of &minus, S responses and by highlighting the effects of &minus, S on Pi uptake, transport, and homeostasis.

Sleep is an evolutionally conserved behavior from vertebrates to invertebrates. The molecular mechanisms that determine daily sleep amounts and the neuronal substrates for homeostatic sleep need remain unknown. Through a large-scale forward genetic screen of sleep behaviors in mice, we previously demonstrated that the Sleepy mutant allele of the Sik3 protein kinase gene markedly increases daily nonrapid-eye movement sleep (NREMS) amounts and sleep need. The Sleepy mutation deletes the in-frame exon 13 encoding a peptide stretch encompassing S551, a known PKA recognition site in SIK3. Here, we demonstrate that single amino acid changes at SIK3 S551 (S551A and S551D) reproduce the hypersomnia phenotype of the Sleepy mutant mice. These mice exhibit increased NREMS amounts and inherently increased sleep need, the latter demonstrated by increased duration of individual NREMS episodes and higher EEG slow-wave activity during NREMS. At the molecular level, deletion or mutation at SIK3 S551 reduces PKA recognition and abolishes 14-3-3 binding. Our results suggest that the evolutionally conserved S551 of SIK3 mediates, together with PKA and 14-3-3, the intracellular signaling crucial for the regulation of daily sleep amounts and sleep need at the organismal level.

Study objectives In humans and other mammals, sleep is altered during pregnancy. However, no studies have been conducted on sleep/wakefulness during pregnancy in mice. In this study, we examined sleep/wakefulness in female C57BL/6 mice during pregnancy. We also examined sleep/wake behaviors in an animal model of preeclampsia, pregnancy-associated hypertensive (PAH) mice, in which increased angiotensin causes hypertension. Methods Sleep/wake behaviors of female C57BL/6 and PAH mice were examined based on electroencephalogram (EEG) or electromyogram recordings before, during, and after pregnancy. To examine whether high blood pressure disrupts the integrity of the blood-brain barrier in PAH mice, Evans blue dye was injected intravenously. Angiotensin II receptor blocker (olmesartan)-administered PAH mice and female Tsukuba hypertensive mice were also examined. Results C57BL/6 mice showed a decreased total wake time and increased nonrapid eye movement (NREM) sleep time during late pregnancy. Rapid eye movement (REM) sleep time did not change during the course of pregnancy. PAH mice exhibited a general slowing of EEG during late pregnancy and subsequently returned to apparently normal sleep/wakefulness after delivery. All PAH mice exhibited multiple focal leakages of Evans blue dye in the brain. Spike-and-wave discharges were observed in 50% of PAH mice. Olmesartan-administered PAH mice did not show general slowing of EEG. Tsukuba hypertensive mice showed a normal time spent in wakefulness and NREM sleep and a decreased total REM sleep time. Conclusions This study showed pregnant-stage-specific changes in sleep/wakefulness in C57BL/6 mice. Furthermore, PAH mice may be useful as an animal model for eclampsia.

Plant purple acid phosphatases (PAPs) belong to a relatively large gene family whose individual functions are poorly understood. Three PAP isozymes that are up-regulated in the cell walls of phosphate (Pi)-starved (-Pi) Arabidopsis thaliana suspension cells were purified and identified by MS as AtPAP12 (At2g27190), AtPAP25 (At4g36350) and AtPAP26 (At5g34850). AtPAP12 and AtPAP26 were previously isolated from the culture medium of -Pi cell cultures, and shown to be secreted by roots of Arabidopsis seedlings to facilitate Pi scavenging from soil-localized organophosphates. AtPAP25 exists as a 55 kDa monomer containing complex NX(S/T) glycosylation motifs at Asn172, Asn367 and Asn424. Transcript profiling and immunoblotting with anti-AtPAP25 immune serum indicated that AtPAP25 is exclusively synthesized under -Pi conditions. Coupled with potent mixed-type inhibition of AtPAP25 by Pi (I50 = 50 μm), this indicates a tight feedback control by Pi that prevents AtPAP25 from being synthesized or functioning as a phosphatase except when Pi levels are quite low. Promoter-GUS reporter assays revealed AtPAP25 expression in shoot vascular tissue of -Pi plants. Development of an atpap25 T-DNA insertion mutant was arrested during cultivation on soil lacking soluble Pi, but rescued upon Pi fertilization or complementation with AtPAP25. Transcript profiling by quantitative RT-PCR indicated that Pi starvation signaling was attenuated in the atpap25 mutant. AtPAP25 exhibited near-optimal phosphatase activity with several phosphoproteins and phosphoamino acids as substrates. We hypothesize that AtPAP25 plays a key signaling role during Pi deprivation by functioning as a phosphoprotein phosphatase rather than as a non-specific scavenger of Pi from extracellular P-monoesters.

Sleep is a behavior conserved from invertebrates to vertebrates, and tightly regulated in a homeostatic manner. The molecular and cellular mechanism determining the amount of rapid eye movement sleep (REMS) and non-REMS (NREMS) remains unknown. Here we identified two dominant mutations affecting sleep/wakefulness through an electroencephalogram/electromyogram-based screening of randomly mutagenized mice. A splicing mutation of the Sik3 protein kinase gene causes a profound decrease in total wake time, due to an increase in inherent sleep need. Sleep deprivation affects regulatory-site phosphorylation of the kinase. Sik3 orthologues regulate sleep also in fruit flies and roundworms. A missense mutation of the leak cation channel NALCN reduces the total amount and episode duration of REMS, apparently by increasing the excitability of REMS-inhibiting neurons. Our results substantiate the utility of forward genetic approach for sleep behaviors in mice, demonstrating the role of SIK3 and NALCN in regulating the amount of NREMS and REMS, respectively.

The discovery of leptin substantiated the usefulness of a forward genetic approach in elucidating the molecular network regulating energy metabolism. However, no successful dominant screening for obesity has been reported, which may be due to the influence of quantitative trait loci between the screening and counter strains and the low fertility of obese mice. Here, we performed a dominant screening for obesity using C57BL/6 substrains, C57BL/6J and C57BL/6N, with the routine use of in vitro fertilization. The screening of more than 5000 mutagenized mice established two obese pedigrees in which single nucleotide substitutions in Mc4r and Sim1 genes were identified through whole-exome sequencing. The mutation in the Mc4r gene produces a premature stop codon and the mutant SIM1 protein lacks transcriptional activity, showing that the haploinsufficiency of SIM1 and MC4R results in obesity. We further examined the hypothalamic neuropeptide expressions in the mutant pedigrees and mice with diet-induced obesity, which showed that each obesity mouse model has distinct neuropeptide expression profiles. This forward genetic screening scheme is useful and applicable to any research field in which mouse models work.

International audience; Phosphate (Pi) is a macronutrient that is essential for plant life. Several regulatory components involved in Pi homeostasis have been identified, revealing a very high complexity at the cellular and subcellular levels. Determining the Pi content in plants is crucial to understanding this regulation, and short real-time(33)Pi uptake imaging experiments have shown Pi movement to be highly dynamic. Furthermore, gene modulation by Pi is finely controlled by localization of this ion at the tissue as well as the cellular and subcellular levels. Deciphering these regulations requires access to and quantification of the Pi pool in the various plant compartments. This review presents the different techniques available to measure, visualize and trace Pi in plants, with a discussion of the future prospects.

The root cap has a fundamental role in sensing environmental cues as well as regulating root growth via altered meristem activity. Despite this well-established role in the control of developmental processes in roots, the root cap’s function in nutrition remains obscure. Here, we uncover its role in phosphate nutrition by targeted cellular inactivation or phosphate transport complementation in Arabidopsis, using a transactivation strategy with an innovative high-resolution real-time 33P imaging technique. Remarkably, the diminutive size of the root cap cells at the root-to-soil exchange surface accounts for a significant amount of the total seedling phosphate uptake (approximately 20%). This level of Pi absorption is sufficient for shoot biomass production (up to a 180% gain in soil), as well as repression of Pi starvation-induced genes. These results extend our understanding of this important tissue from its previously described roles in environmental perception to novel functions in mineral nutrition and homeostasis control. DOI: http://dx.doi.org/10.7554/eLife.14577.001, eLife digest All plants need phosphate to grow because it is a major component of DNA and many other biological molecules. Most of the Earth’s soil is poor in phosphate, and so farmland is routinely supplemented with fertilizers to provide crops with this essential nutrient. However, phosphate fertilizers are becoming scarce and their quality is expected to decline in the near future. Plant biologists must therefore determine how to adapt plants to a restricted supply of this resource, in order to sustain high crop yields for the growing world population. Plants are known to absorb phosphate through specific protein-based transporters located in the cells that make up the outer layer of roots. These proteins are highly concentrated at the root tip, and while this specialized tissue is well-known for perceiving gravity and light, it had not been shown to play a role in phosphate absorption. Kanno, Arrighi et al. have now used genetically modified Arabidopsis plants to demonstrate that phosphate can be taken up via the small cells that surround the root tip. The experiments showed that the absorbed phosphate rapidly reaches the leaves within minutes, helps the plant grow and modifies its metabolism. As the root tip can accumulate high amounts of phosphate in order to sustain its own activity, it was important to distinguish uptake of phosphate from the environment from redistribution of phosphate already within the plant. Kanno, Arrighi et al. tackled this issue through the development of a new radioactive micro-imaging technique. Phosphate transporters are also present within the cell layers within the root, but their purpose and activity are not well described. Further studies are needed to analyze the role of other root cell layers in phosphate uptake and transport, and the newly developed techniques will help decipher the mechanisms involved. DOI: http://dx.doi.org/10.7554/eLife.14577.002

A new real-time radioisotope imaging system (RRIS) to study the kinetics of nutrient uptake and transfer of photosynthetic products in a living plant was developed and evaluated through a test run. 14C is a common radioisotope of carbon and useful to trace the photosynthetic products as well as a low energy beta emitter. The rationale of this study was to develop a RRIS that has the ability to detect low energy beta emitters, such as 14C, 35S, and 45Ca. To achieve compatibility between the detection of low energy beta emitters and irradiation of the test plant, an intermittent lighting system was added to the RRIS. Furthermore, a commercially available digital camera was added to the RRIS for acquisition of photographic images of the test plants. The capabilities of the new RRIS were evaluated through a test run by using seedlings of rice plants and 35S-labeled sulfate. It was shown that the new RRIS was able to detect 35S absorbed by rice plant seedlings, and it was able to acquire photon-counting images and photographic images of the test plants simultaneously. Despite some limitations, the new RRIS provides a means to study the kinetics of elements in plants by utilizing low energy beta emitters.

Ionic nutrition is essential for plant development. Many techniques have been developed to image and (or) measure ionic movement in plants. Nevertheless, most of them are destructive and limit the analysis. Here, we present the development of radioisotope imaging techniques that overcome such restrictions and allow for real-time imaging of ionic movement. The first system, called macroimaging, was developed to visualize and measure ion uptake and translocation between organs at a whole-plant scale. Such a device is fully compatible with illumination of the sample. We also modified fluorescent microscopes to set up various solutions for ion uptake analysis at the microscopic level. Both systems allow numerical analysis of images and possess a wide dynamic range of detection because they are based on radioactivity.

リアルタイムRIイメージングシステムを搭載したラジオアイソトープ顕微鏡(VIM-micro)を用いて，植物の根における55Feの動態を解析した。VIM-microの55Feに対する検出感度はイメージングプレートよりも顕著に高く，2分間の積算により，根における55Feの分布を可視化できた。10kBq/μLの55Feをシロイヌナズナとイネの根の下部側に処理し，その後2時間にわたって60枚の根の画像を取得することで55Feの動態を解析した。いずれの植物においても，55Feは根の先端部に優先的に分配された。阻害剤の処理，あるいは煮沸によって不活性化した根を，通常の根と比較解析したところ，30分以降に見られる55Feの根先端部への蓄積が，生物活性によるものであることが判明した。これらの結果により，Feは，重要なシンク器官である根端部に積極的に輸送されることが示された。

It is very important to visualize the process of nutrient absorption and distribution to study the physiological activity of the plant. We developed a real-time radioisotope (RI) imaging system, where RI tracers were applied to the plant sample. This system allowed the quantitative measurement concerning the uptake of nutrients labeled with radioisotopes, such as 45Ca, 35S, 32P and 14C as long as several days. The β-rays emitted from the sample were converted to light by a CsI(Tl) scintillator and were guided to a highly sensitive CCD camera. The scintillator surface was covered with an Al plate to avoid LED light penetration but allow selected β-ray penetration. We employed Lotus japonicus for the plant sample and observed the 32P-phosphate absorption in roots and the accumulation to the aboveground part of the plant. The environment condition of daytime and night was simulated by the ON/OFF of LED timer and the accumulation manner of the 32P-phosphate in roots and leaves during daytime and night was analyzed. The accumulation of 32P-phosphate in leaves was highly dependant on light irradiation and higher when the LEDs was turned on, whereas the absorption of 32P-phosphate in root was higher when the LEDs was turned off. The transfer function concerning the transportation of phosphate within the plant during the developmental stage was obtained from the analysis of 32P uptake images. We are now trying to get specific moving images of each radioisotope when two kinds of isotopes, such as 32P and 35S, were applied at the same time to the plant, through an image analysis.

We present double-tracer autoradiography with 35S and 32P using imaging plates and an aluminum plate to visualize the individual distribution of sulfur and phosphorus in Brassica napus L.. Because of the different attenuation efficiencies of β-rays emitted from 35S and 32P, the specific image of 32P was taken when an aluminum plate was placed between the sample and the imaging plate. The specific image of 35S was obtained by subtracting the 32P image from the 35S and 32P image. It was observed that high amount of 35S was distributed at the edge of the leaves and in root tips. The 32P was distributed throughout the whole plant and it was found that the phosphorous amount was higher at the shoot apical meristems than those in leaves.

In this study, real-time imaging using γ-emitting nuclide, 109Cd, was studied and sensitivity and resolution of the system we developed was evaluated. When RI was applied to the plant, the radiation from the RI distributed throughout the plant was converted to visible light by a scintillator. Then, this weak light was multiplied by a micro-channel plate(MCP)and photographed by a charge-coupled device(CCD)camera. 109Cd emits 88keV γ-rays together with 22keV X-rays, and therefore it is necessary to investigate effective condition for designing an observation system for the measurement of radiation from 109Cd. Analysis was performed to determine which rays, γ-rays or X-rays, were mainly detected by this system and the optimal scintillator thickness for imaging 109Cd was investigated. Then the sensitivity and resolution of the system was evaluated.

We present the real-time RI imaging and analyzing system to study the kinetics of nutrient uptake manner in a living plant. The system allowed light condition for the up-ground part of the plant and continuous dark condition for the root part, therefore, light/dark cycles was set as 16/8 h. There was 9,000 lx of LED lights in an aluminum container where the plant was set. The container was shielded well so that there was no light leakage to damage highly sensitive CCD camera which detected beta-rays from the sample. With this system, RI imaging was able to perform for 6 days without damaging the activity of the plant.

リン酸は，植物のリン酸栄養環境応答時の認識機構に関わる重要な因子である。そこで，植物体内のリン酸吸収移行の特徴について知るためにリン酸欠乏下のダイズを用い32Pトレーサ実験を行った。トレーサ実験では，従来のImaging Plateと，リン酸の速い動きを定量的にイメージングできるリアルタイムRIイメージングシステムを用いた。その結果，リン酸は通常条件で栽培した場合には常に新葉への移行が古い葉への移行よりも約3倍高くなるのに対して，リン酸欠乏環境下では古い葉へも積極的に移行することが示された。

For combination with microscope, we developed real-time autoradiography system for micro-scale analysis with adjustment of the CsI(Ti) scintillator thickness for higher resolution and applying tapered fiber optic plate for magnification of autoradiograph image. We combined real-time autoradiography system with an inverted fluorescent microscope so that an autoradiograph image as well as fluorescent image, bright-field image can be acquired at the same time. In the case of observation of sliced soybean stalk traced 45CaCl, the fluorescent and bright-field image was acquired which magnified to 50 times, the autoradiograph image of 45Ca distribution in the tissue was acquired in almost same scale. The new microscopic system which can acquire autoradiograph image of labeled signals (low molecular weight) is expected to develop the signal transduction study and gene expression, combined with fluorescent protein techniques such as GFP etc.

Real-time imaging analysis in a living plant allows studying the mechanism of chemical transport or signal transduction more in detail. We present the real-time autoradiography system to analyze the movement of β-ray emitters in a living plant. The system was consisted of a FOS (a fiber optic plate deposited with CsI(Ti) scintillator) and a GaAsP imaging intensifier unit. Though the resolution is about the same as that of an imaging plate(IP), the sensitivity was more than 10 times higher than that of an IP. Using this system, 32P movement in a young leaf of soybean was analyzed when 32P was supplied from the root.

We developed a quantitative, real-time imaging system of labeled compounds in a living plant. The system was composed of CsI scintillator to convert β-rays to visible light and an image intensifier unit (composed of GaAsP semiconductor and MCP; micro channel plate) to detect extremely weak light. When the sensitivity and resolution of the image of our system was compared with that of an imaging plate (IP), the sensitivity of our system (with 20 minutes) was higher than that of an IP, with similar quality to that of an IP. Using this system, the translocation of 32P in a soybean plant tissue was shown in successive images.

Soil phosphate represents the only source of phosphorus for plants and, consequently, is its entry into the trophic chain. This major component of nucleic acids, phospholipids, and energy currency of the cell (ATP) can limit plant growth because of its low mobility in soil. As a result, root responses to low phosphate favor the exploration of the shallower part of the soil, where phosphate tends to be more abundant, a strategy described as topsoil foraging. We will review the diverse developmental strategies that can be observed among plants by detailing the effect of phosphate deficiency on primary and lateral roots. We also discuss the formation of cluster roots: an advanced adaptive strategy to cope with low phosphate availability observed in a limited number of species. Finally, we will put this work into perspective for future research directions.

The main source of phosphorus for plants is inorganic phosphate (Pi), which is characterized by its poor availability and low mobility. Uptake of this element from the soil relies heavily upon the PHT1 transporters, a specific family of plant plasma membrane proteins that were identified by homology with the yeast PHO84 Pi transporter. Since the discovery of PHT1 transporters in 1996, various studies have revealed that their function is controlled by a highly complex network of regulation. This review will summarize the current state of research on plant PHT1 multigenic families, including physiological, biochemical, molecular, cellular, and genetics studies.