Dynamic Changes in the Distribution of Minerals in Relation to Phytic Acid Accumulation during Rice Seed Development

Phytic acid (inositol hexakisphosphate [InsP₆]) is the storage compound of phosphorus in seeds. As phytic acid binds strongly to metallic cations, it also acts as a storage compound of metals. To understand the mechanisms underlying metal accumulation and localization in relation to phytic acid storage, we applied synchrotron-based x-ray microfluorescence imaging analysis to characterize the simultaneous subcellular distribution of some mineral elements (phosphorus, calcium, potassium, iron, zinc, and copper) in immature and mature rice (Oryza sativa) seeds. This fine-imaging method can reveal whether these elements colocalize. We also determined their accumulation patterns and the changes in phosphate and InsP₆ contents during seed development. While the InsP₆ content in the outer parts of seeds rapidly increased during seed development, the phosphate contents of both the outer and inner parts of seeds remained low. Phosphorus, calcium, potassium, and iron were most abundant in the aleurone layer, and they colocalized throughout seed development. Zinc was broadly distributed from the aleurone layer to the inner endosperm. Copper localized outside the aleurone layer and did not colocalize with phosphorus. From these results, we suggest that phosphorus translocated from source organs was immediately converted to InsP₆ and accumulated in aleurone layer cells and that calcium, potassium, and iron accumulated as phytic acid salt (phytate) in the aleurone layer, whereas zinc bound loosely to InsP₆ and accumulated not only in phytate but also in another storage form. Copper accumulated in the endosperm and may exhibit a storage form other than phytate.The transport of nutrients into developing seeds has received considerable attention. During the grain-filling stage, plants remobilize and transport nutrients distributed throughout the vegetative source organs into seeds. Plant seeds contain large amounts of phosphorus (P) in organic form, which supports growth during the early stages of seedling development. Most of the P in seeds is stored in the form of phytic acid (inositol hexakisphosphate [InsP₆]). Seeds also accumulate mineral nutrients such as potassium (K), magnesium (Mg), calcium (Ca), iron (Fe), zinc (Zn), copper (Cu), and manganese (Mn), which are used in seedling growth. Phytic acid acts as a strong chelator of metal cations and binds them to form phytate, a salt of InsP₆ (Lott et al., 2002; Raboy, 2009). During germination, phytate is decationized and hydrolyzed by phytases, and then inorganic phosphates, inositol, and various minerals are released from the phytate (Loewus and Murthy, 2000). Phytate accumulates within protein bodies, generally of vacuolar origin, in seed storage cells and is usually concentrated in spherical inclusions called globoids. Many studies of the elemental composition of phytate in seeds have been published. Energy-dispersive x-ray microanalyses of many plant species have revealed that, other than P, globoids contain mainly K and Mg as well as low levels of Ca, Mn, Fe, and Zn (Lott, 1984; Lott et al., 1995; Wada and Lott, 1997). This indicates that phytate is a mixed salt of these cations.Whether all storage metal elements can bind equally to InsP₆ is not known, although most elements are thought to exist in seeds in the form of phytate (Raboy, 2009). To form phytate, P and the other elements must be present in the same place. Therefore, determination of the precise locations of P and other elements in seed tissues makes it possible to judge whether an element exists in the form of phytate. Differences in metal distribution with P might suggest a storage form other than phytate. For determining distributions, synchrotron-based x-ray microfluorescence (µ-XRF) imaging utilizing an x-ray microbeam is a powerful tool. The microbeam excites the elements, thereby revealing the details of their spatial distribution. The development of focusing optics for high-energy x-rays using a Kirkpatrick-Baez mirror raises the imaging resolution of elements in µ-XRF analysis. A focal spot size smaller than 1 µm with x-ray energy as high as 100 keV enables detection of the subcellular distribution of elements in plant tissues (Fukuda et al., 2008; Takahashi et al., 2009).Whether there is an order in the affinity of elements for phytic acid in plant cells remains unknown. The stability of InsP₆-metal complexes has been estimated by in vitro titration (Maddaiah et al., 1964; Vohra et al., 1965; Persson et al., 1998). The binding strength of InsP₆ with metal is stronger for Zn and Cu than for Fe, Mn, and Ca. We also do not know if the mineral composition of phytate in seeds is determined by the relative abundance of these elements in the seed or by their biochemical characteristics. As a first step to address these issues, we examined the simultaneous changes in the distribution of P and metal elements during seed development using µ-XRF imaging analysis.Our objective in this study was to observe the dynamic changes in the distribution of some nutritionally important minerals (P, Ca, K, Fe, Zn, and Cu) in relation to the accumulation of phytic acid during rice (Oryza sativa) seed development.     

Neural correlates of the difference between working memory speed and simple sensorimotor speed: an fMRI study

The difference between the speed of simple cognitive processes and the speed of complex cognitive processes has various psychological correlates. However, the neural correlates of this difference have not yet been investigated. In this study, we focused on working memory (WM) for typical complex cognitive processes. Functional magnetic resonance imaging data were acquired during the performance of an N-back task, which is a measure of WM for typical complex cognitive processes. In our N-back task, task speed and memory load were varied to identify the neural correlates responsible for the difference between the speed of simple cognitive processes (estimated from the 0-back task) and the speed of WM. Our findings showed that this difference was characterized by the increased activation in the right dorsolateral prefrontal cortex (DLPFC) and the increased functional interaction between the right DLPFC and right superior parietal lobe. Furthermore, the local gray matter volume of the right DLPFC was correlated with participants' accuracy during fast WM tasks, which in turn correlated with a psychometric measure of participants' intelligence. Our findings indicate that the right DLPFC and its related network are responsible for the execution of the fast cognitive processes involved in WM. Identified neural bases may underlie the psychometric differences between the speed with which subjects perform simple cognitive tasks and the speed with which subjects perform more complex cognitive tasks, and explain the previous traditional psychological findings.     

Effects of the Higashi-Nihon earthquake: posttraumatic stress, psychological changes, and cortisol levels of survivors

On March 11, 2011, the Pacific side of Japan's northeast was devastated by an earthquake and tsunami. For years, many researchers have been working on ways of examining the psychological effects of earthquakes on survivors in disaster areas who have experienced aftershocks, catastrophic fires, and other damage caused by the earthquake. The goal of this study is to examine scores on psychological measures and salivary cortisol level in these individuals both before and three months after the earthquake. The participants had been measured for these variables before the earthquake. After the earthquake, we carried out PTSD screening using CAPS for participants for another experiment, and then again conducted the aforementioned tests. We collected saliva samples from all survivors. Our results show that social relationship scores on the WHO-QOL26, negative mood scores of the WHO-SUBI, total GHQ score, POMS confusion scores, and CMI emotional status score after the earthquake showed scores indicating significantly decreased compared to before the earthquake. On the other hand, salivary cortisol levels after the earthquake was significantly increased compared to before the earthquake. Moreover, the result of a multiple regression analysis found that negative mood score on the WHO-SUBI and social relationship score on the WHO-QOL26 were significantly related to salivary cortisol levels. Our results thus demonstrate that several psychological stress induced by the earthquake was associated with an increase in salivary cortisol levels. These results show similar findings to previous study. We anticipate that this study will provide a better understanding of posttraumatic responses in the early stages of adaptation to the trauma and expand effective prevention strategies and countermeasures for PTSD.     

An inducible RNA interference system in Physcomitrella patens reveals a dominant role of augmin in phragmoplast microtubule generation

Mitosis is a fundamental process of eukaryotic cell proliferation. However, the molecular mechanisms underlying mitosis remain poorly understood in plants partly because of the lack of an appropriate model cell system in which loss-of-function analyses can be easily combined with high-resolution microscopy. Here, we developed an inducible RNA interference (RNAi) system and three-dimensional time-lapse confocal microscopy in the moss Physcomitrella patens that allowed in-depth phenotype characterization of the moss genes essential for cell division. We applied this technique to two microtubule regulators, augmin and γ-tubulin complexes, whose mitotic roles remain obscure in plant cells. Live imaging of caulonemal cells showed that they proceed through mitosis with continual generation and self-organization of acentrosomal microtubules. We demonstrated that augmin plays an important role in γ-tubulin localization and microtubule generation from prometaphase to cytokinesis. Most evidently, microtubule formation in phragmoplasts was severely compromised after RNAi knockdown of an augmin subunit, leading to incomplete expansion of phragmoplasts and cytokinesis failure. Knockdown of the γ-tubulin complex affected microtubule formation throughout mitosis. We conclude that postanaphase microtubule generation is predominantly stimulated by the augmin/γ-tubulin machinery in moss and further propose that this RNAi system serves as a powerful tool to dissect the molecular mechanisms underlying mitosis in land plants.     

Toll-like receptor 5 forms asymmetric dimers in the absence of flagellin

The structure of full-length human TLR5 determined by electron microscopy single-particle image reconstruction at 26 Å resolution shows that TLR5 forms an asymmetric homodimer via ectodomain interactions. The structure shows that like TLR9, TLR5 dimerizes in the absence of ligand. The asymmetry of the dimer suggests that TLR5 may recognize two flagellin molecules cooperatively to establish an optimal flagellin response threshold. A TLR5 homology model was generated and fitted into the electron microscopy structure. All seven predicted N-linked glycosylation sites are exposed on the molecular surface, away from the dimer interface. Glycosylation at the first five sites was confirmed by tandem mass spectrometry. Two aspartate residues proposed to interact with flagellin (Asp294 and Asp366) are sterically occluded by a glycan at position 342. In contrast, the central region of the ectodomains near the dimer interface is unobstructed by glycans. Ligand binding in this region would be consistent with the ligand binding sites of other TLRs.     

Mechanism of case processing in the brain: an FMRI study

In sentence comprehension research, the case system, which is one of the subsystems of the language processing system, has been assumed to play a crucial role in signifying relationships in sentences between noun phrases (NPs) and other elements, such as verbs, prepositions, nouns, and tense. However, so far, less attention has been paid to the question of how cases are processed in our brain. To this end, the current study used fMRI and scanned the brain activity of 15 native English speakers during an English-case processing task. The results showed that, while the processing of all cases activates the left inferior frontal gyrus and posterior part of the middle temporal gyrus, genitive case processing activates these two regions more than nominative and accusative case processing. Since the effect of the difference in behavioral performance among these three cases is excluded from brain activation data, the observed different brain activations would be due to the different processing patterns among the cases, indicating that cases are processed differently in our brains. The different brain activations between genitive case processing and nominative/accusative case processing may be due to the difference in structural complexity between them.     

Interaction and stoichiometry of the peripheral stalk subunits NtpE and NtpF and the N-terminal hydrophilic domain of NtpI of Enterococcus hirae V-ATPase

The vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain and an integral membrane domain connected by a central stalk and a few peripheral stalks. The number and arrangement of the peripheral stalk subunits remain controversial. The peripheral stalk of Na+-translocating V-ATPase from Enterococcus hirae is likely to be composed of NtpE and NtpF (corresponding to subunit G of eukaryotic V-ATPase) subunits together with the N-terminal hydrophilic domain of NtpI (corresponding to subunit a of eukaryotic V-ATPase). Here we purified NtpE, NtpF, and the N-terminal hydrophilic domain of NtpI (NtpI(Nterm)) as separate recombinant His-tagged proteins and examined interactions between these three subunits by pulldown assay using one tagged subunit, CD spectroscopy, surface plasmon resonance, and analytical ultracentrifugation. NtpI(Nterm) directly bound NtpF, but not NtpE. NtpE bound NtpF tightly. NtpI(Nterm) bound the NtpE-F complex stronger than NtpF only, suggesting that NtpE increases the binding affinity between NtpI(Nterm) and NtpF. Purified NtpE-F-I(Nterm) complex appeared to be monodisperse, and the molecular masses estimated from analytical ultracentrifugation and small-angle x-ray scattering (SAXS) indicated that the ternary complex is formed with a 1:1:1 stoichiometry. A low resolution structure model of the complex produced from the SAXS data showed an elongated "L" shape.     

Mechanism of robust circadian oscillation of KaiC phosphorylation in vitro

By incubating the mixture of three cyanobacterial proteins, KaiA, KaiB, and KaiC, with ATP in vitro, T. Kondo and his colleagues in recent work reconstituted the robust circadian rhythm of the phosphorylation level of KaiC. This finding indicates that protein-protein interactions and the associated hydrolysis of ATP suffice to generate the circadian rhythm. Several theoretical models have been proposed to explain the rhythm generated in this “protein-only” system, but the clear criterion to discern different possible mechanisms was not known. In this article, we discuss a model based on two basic assumptions: the assumption of the allosteric transition of a KaiC hexamer and the assumption of the monomer exchange between KaiC hexamers. The model shows a stable rhythmic oscillation of the phosphorylation level of KaiC, which is robust against changes in concentration of Kai proteins. We show that this robustness gives a clue to distinguish different possible mechanisms. We also discuss the robustness of oscillation against the change in the system size. Behaviors of the system with the cellular or subcellular size should shed light on the role of the protein-protein interactions in in vivo circadian oscillation.